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Rapid and Precise Engineering of the Caenorhabditis elegans Genome with Lethal Mutation Co-Conversion and Inactivation of NHEJ Repair

As in other organisms, CRISPR/Cas9 methods provide a powerful approach for genome editing in the nematode Caenorhabditis elegans. Oligonucleotides are excellent repair templates for introducing substitutions and short insertions, as they are cost effective, require no cloning, and appear in other organisms to target changes by homologous recombination at DNA double-strand breaks (DSBs). Here, I describe a methodology in C. elegans to efficiently knock in epitope tags in 8–9 days, using a temperature-sensitive lethal mutation in the pha-1 gene as a co-conversion marker. I demonstrate that 60mer oligos with 29 bp of homology drive efficient knock-in of point mutations, and that disabling nonhomologous end joining by RNAi inactivation of the cku-80 gene significantly improves knock-in efficiency. Homology arms of 35–80 bp are sufficient for efficient editing and DSBs up to 54 bp away from the insertion site produced knock-ins. These findings will likely be applicable for a range of genome editing approaches in C. elegans, which will improve editing efficiency and minimize screening efforts.

S EQUENCE-SPECIFIC nucleases are a critical tool for manipulation of DNA sequences. The bacterial type II clustered regularly interspaced short palindromic repeats (CRISPR) system, which normally protects against viral DNA and provides a memory of exposure (Jinek et al. 2012), has recently revolutionized genome editing in multiple organisms (Cong et al. 2013;DiCarlo et al. 2013;Gratz et al. 2013;Hwang et al. 2013;Li et al. 2013;Ran et al. 2013;Gratz et al. 2014;Nakanishi et al. 2014). For genome editing, the system has been simplified to two components: the Cas9 nuclease, which generates DNA double-strand breaks (DSBs), and a chimeric small guide RNA (sgRNA) that fills the function of two small RNAs in the native bacterial system (Cong et al. 2013). Specific genomic sequences are targeted by the 59-most 15-20 bp of the sgRNA through the formation of an RNA:DNA hybrid (Jinek et al. 2012;Mali et al. 2013). An NGG motif (protospacer adjacent motif, PAM) must immediately follow the target sequence in the genome (Jinek et al. 2012;Ran et al. 2013). This PAM directs Cas9 to cleave the DNA 3 bp 59 to the PAM (Jinek et al. 2012). Depending on the desired experimental outcome, one can select for error-prone repair by pathways such as nonhomologous end joining (NHEJ) to generate insertion-deletion (indel) mutations, or homologous recombination to knock in specific sequences.
Initial genome editing methods in Caenorhabditis elegans harnessed excision of a Tc or Mos transposon to generate a DSB, and a plasmid repair template to knock in (Plasterk and Groenen 1992;Robert and Bessereau 2007;Frøkjaer-Jensen et al. 2008;Frøkjaer-Jensen et al. 2012), or delete (Frøkjaer-Jensen et al. 2010) desired sequences through homologous recombination. These methods are robust, but the relative rarity of the editing event requires use of a selectable marker, such as unc-119 rescue or antibiotic resistance (Frøkjaer-Jensen et al. 2008;Giordano-Santini et al. 2010), and a transposon site is ideally needed within 1-2 kb of the desired edit (Robert and Bessereau 2007). Zinc finger and transcription activator-like effector nucleases (Wood et al. 2011) and CRISPR/Cas9 have allowed for similar efficient editing without the constraint of transposon insertions. In particular, the ease and rapidity of generating new sgRNAs for the CRISPR/Cas9 system means that transgenic strains can be created precisely and rapidly and any endogenous NGG sequence can theoretically be targeted. Several CRISPR/ Cas9 systems have been described, each with individual strengths and weaknesses (Waaijers and Boxem 2014). Cas9 can be delivered by micro-injection of in vitro transcribed mRNA (Chiu et al. 2013;Lo et al. 2013), pure protein (Cho et al. 2013), or plasmid DNA (Chen et al. 2013b;Dickinson et al. 2013;Friedland et al. 2013;Katic and Grosshans 2013;Waaijers et al. 2013). Similarly, the sgRNAs can be introduced by in vitro transcription, which does not require polyA tailing or 59 methyl cap addition (Chiu et al. 2013;Cho et al. 2013;Lo et al. 2013), or driven by RNA polymerase III promoters such as U6 (Chen et al. 2013b;Dickinson et al. 2013;Friedland et al. 2013;Katic and Grosshans 2013;Waaijers et al. 2013) or rpr-1 (Chiu et al. 2013). Most groups use the chimeric sgRNA, though a previous report described higher in vitro nuclease activity using the two separate bacterial small RNAs (Lo et al. 2013). Multiple groups have developed protocols for both knockouts and knock-ins. Knock-ins have been primarily generated through efficient selection schemes based on the earlier MosI-mediated single-copy transgene insertion methods using genetic markers such as unc-119 (Dickinson et al. 2013), drug resistance markers (Chen et al. 2013b), or fluorescence . Typically, plasmid repair templates with 1 kb or more of homology flanking the insert have been used (Chen et al. 2013b;Dickinson et al. 2013;Tzur et al. 2013;. Recently, several reports have described methods to introduce single-basepair changes, small epitopes, and larger tags such as GFP without the need for selectable markers; these approaches either directly screened all F 1 progeny from co-injection marker positive animals (Paix et al. 2014) or employed a co-CRISPR/ co-conversion approach where selection for one editing event resulted in an enrichment for edits at unrelated loci (Arribere et al. 2014;. Direct screening of F 1 's allows editing without introduction of additional mutations, but is more labor intensive, while co-CRISPR/co-conversion allows for identification of editing events while minimizing hands-on screening, but requires outcrossing or meiotic segregation of the marker allele. Co-CRISPR selects for mutation in the unc-22 gene, with mutant homozygotes identified in the F 2 progeny of co-injection marker positive animals, or less frequently in the F 1 progeny , though haploinsufficiency of the unc-22 locus in 1% nicotine should allow for identification of unc-22 mutant heterozygotes in F 1 animals (Moerman and Baillie 1979). Coconversion selects for knock-in of dominant alleles in the dpy-10, sqt-1, or rol-6 genes in the F 1 progeny of injected animals (Arribere et al. 2014).
The goal of this study was to test whether repair of a temperature-sensitive lethal point mutation could be used as an alternate co-conversion marker, as such an approach could in theory provide robust selection, minimal screening, and no requirement for outcrossing or meiotic segregation of marker alleles/mutations. I focused on using single-stranded oligonucleotides (oligos) as a template as they have been successfully used in a range of model organisms (Igoucheva et al. 2001;Storici et al. 2003;Chen et al. 2011;Bedell et al. 2012;DiCarlo et al. 2013), are cost effective, and require no cloning. In C. elegans, oligonucleotides have been used to introduce single-base changes (Arribere et al. 2014;, inactivate genes by introducing premature stop codons or deleting sequences (Lo et al. 2013;Paix et al. 2014), or insert small protein epitopes (Lo et al. 2013;Paix et al. 2014). Introduction of epitopes seamlessly into endogenous loci has numerous experimental uses: chromatin immunoprecipitation, purification of protein complexes followed by mass spectrometry, and detection of proteins by immunofluorescence or immunoblotting. Furthermore, optimizing insertion of epitope tags using oligonucleotide templates is almost certain to be applicable to single-base editing. Here, I describe a robust, cost effective, widely applicable method using pha-1 coconversion and inactivation of NHEJ repair to rapidly and precisely engineer the C. elegans genome using oligo-templated repair with the CRISPR/Cas9 system.

Genetics
The following strains were used in this study: N2(WT), GE24 pha-1(e2123) III, RB873 lig-4(ok716) III, which were provided by the Caenorhabditis Genetics Center. N2 animals and lig-4 mutants were propagated at 20°, while pha-1(e2123) mutants were propagated at 15°. Animals were maintained on nematode growth medium seeded with Escherichia coli OP50 (Brenner 1974). For later injection experiments, pha-1(e2123) mutants were maintained on HB101. Growth conditions for each experiment are indicated in figure and table legends. Strains generated for this study are listed in Supporting Information, File S1, Table S1.

Microinjection
Mixtures of plasmids and oligos were microinjected into the gonad of young adult animals. Plasmids were purified using a Qiagen midiprep kit. Oligos were resuspended in TE buffer and working stocks of 2 mg/ml were made with nucleasefree dH 2 0. Repair template oligos are listed in Table S2. For PCR-generated PU6::sgRNA templates, PCR products were purified from 100-ml reactions and concentrated 10-fold using a DNA Clean and Concentrator kit (Zymo Research, no. D4004).

MfeI deletion assay
Wild-type (WT) animals were microinjected with 50 ng/ml of pJW1138 or pJW1236 [klp-12 targeting CRISPR/Cas9 plasmids with original and flipped plus extended (F+E) sgRNAs, respectively], 10 ng/ml myo-2::tdTomato co-injection marker, and 40 ng/ml of pBluescript DNA. Marker positive F 1 animals were picked into 30 ml of M9 + gelatin in a 96 well. Concentrated OP50 food (30 ml; see the "PCR-based knock-in screening" protocol (File S1) in the for recipe) was then added and F 1 's were incubated for 3-4 days at 25°to allow progeny to develop. Worms were lysed and genotyped as described in the PCR-based knock-in screening section (see Supporting Information). To each 10 ml PCR, 5 ml of MfeI-HF (NEB, R3589L) digestion mixture (1 ml 103 CutSmart buffer, 3.5 ml dH20, 0.5 ml MfeI-HF) was added. The reaction was mixed, incubated at 37°for 1 hr, and then resolved on a 1.5% TAE-agarose gel.
Temperature-sensitive pha-1 co-conversion screening For temperature-sensitive pha-1 [pha-1(ts)] co-conversion experiments, animals were microinjected with 60 ng/ml of pJW1285 (pha-1 targeting) CRISPR/Cas9 plasmid, 60 ng/ml of either pJW1285 (nhr-23 PAM no. 1 targeting), or pJW1268 (nhr-23 PAM no. 2 targeting) Cas9 plasmid and 50 ng/ml of the appropriate repair oligos. In experiments where a co-injection marker was included, myo-2::tdTomato was used at 10 ng/ml. PCR-derived PU6::sgRNA templates were injected at a concentration of 25 ng/ml along with 50 ng/ml of pJW1285 (pha-1 targeting CRISPR/Cas9) and 50 ng/ml of the appropriate repair oligos. Injected adults grown at the permissive temperature (15°) were singled into wells of a 24-well plate containing NGM-lite agar and seeded with OP50, shifted to 25°, and incubated for 3-4 days. Rescued F 1 's (L3s to adults) were the only animals other than the P0 animals observed in wells. These F 1 rescues were singled onto individual plates and incubated for two days to allow progeny development. The parental F 1 animals were then genotyped by restriction digestion, as described below, to identify animals with a 23FLAG insertion. The remaining PCR product was purified using a DNA Clean and Concentrator kit (Zymo Research) and then sequenced using knock-in specific primers (Table S3). To recover homozygotes for sequence-verified knock-ins, 12-24 F 2 progeny were singled onto individual plates, incubated at 25°for 2 days, and the parental F 2 was genotyped to confirm the 23FLAG insertion. These progeny were also genotyped for pha-1(ts) repair by PCR and CEL-1 digestion followed by sequencing of candidate repair homozygotes.

Genotyping PCRs and restriction digestion
Single F 1 animals were picked into 10 ml of single-worm lysis buffer [10 mM Tris (pH 8.3), 50 mM KCl, 2.5 mM MgCl 2 , 0.45% IGEPAL, 0.45% Tween-20] containing 1 mg/ml proteinase K. Tubes were incubated on dry ice for 15 min, 62°for 1 hr, and then heated to 95°for 20 min to inactivate the proteinase K. Genotyping PCRs were performed using Phusion polymerase (NEB, no. M0530S) and High Fidelity buffer; 1/10 volume of lysate was used as template in the PCR. For identification of knock-in events in the F 1 , 30-ml PCRs were performed. Five microliters of this PCR was removed and BamHI digested by adding 10 ml of digestion mix per PCR (1 ml 103 NEBuffer 3, 0.5 ml BamHI, and 8.5 ml dH20) and digesting for 1 hr at 37°b efore resolving on a 1.5% TAE-agarose gel. CEL-1 was purified from four heads of nonorganic celery (Safeway) with the celery juice extracted with a BJE510XL 900W juicer (Breville). Purification was performed as described (Yang et al. 2000) to the end of dialysis in step I, as described by Lo et al. (2013). For CEL-1 digestions, 10 ml of enzyme mix (2 ml CEL-1 + 2 ml 5xPhusion High Fidelity buffer + 6 ml dH20) was added to the 5-ml PCR and incubated at 42°for 1 hr prior to resolving on a 1.5% TAEagarose gel, as described above. Oligos used for genotyping are listed in Table S3.
Generation of U6 promoter::sgRNA templates by PCR The U6 promoter and chimeric sgRNA(F+E) template were amplified from pJW1219 and cloned into the pCR-Blunt II-TOPO vector (Invitrogen, no. K2800-20) to generate pJW1310 and pJW1311, respectively. The U6 promoter was then PCR amplified from pJW1310 with oligos 1787 and 1788, while the sgRNA template was amplified from pJW1311 with oligo 1790 and a target-specific 60mer that contained 20 bp of homology to the U6 promoter, 20 bp of new sgRNA target sequence, and 20 bp of homology to the sgRNA template (Table S3). New sgRNA template primers can be made by replacing the N 20 in the following sequence with 20 bp of target specific sequence: 59-cctcctattgcga gatgtcttg(N 20 )gtttaagagctatgctgg-39. The U6 promoter and sgRNA template PCRs were mixed (0.5 ml each per 100 ml PCR reaction) and amplified using the external primers (1787 and 1790; Table S3). The cycling parameters were: (i) 98°denaturation; (ii) 35 cycles of 98°for 10 sec, 61°for 30 sec, 72°for 20 sec; and (iii) 72°for 1-min final extension. To generate more PU6::sgRNA templates, the fused product was used in a 100-ml nested reaction with primers 1793 and 1794 (Table S3).

Immunoblotting
Lysates were generated as described in File S1 and resolved on Mini-PROTEAN TGX stain-free 4-15% gradient gels . Stain-free gels (Bio-Rad) contain a compound evenly distributed in the precast acrylamide gel that reacts with tryptophan following UV exposure and gives a strong fluorescent signal that can be used to stain for total-protein levels in acrylamide gels, monitor transfer in immunoblotting, and serve as a loading control. Following resolution, the stain-free compound was activated (Posch et al. 2013) and proteins were transferred to an Immobilon FL PVDF membrane (Millipore, no. IPFL00010) at 100 V for 60 min. Total protein, pre-and post-transfer, was monitored using the stain-free fluorophore as described (Posch et al. 2013). Stain-free imaging of total protein was used to confirm equal loading. The blots were sequentially probed with anti-FLAG (1:1000) (M2 clone, Sigma, no. F1504), and sheep antimouse-HRP conjugate (1:5000) (GE Healthcare, no. RPN4201). WesternBright Sirius HRP substrate (Advansta, no. K-12043-C20) and ECL Prime (GE Healthcare, no. RPN2232) were used to develop the blots in Figures 2 and 5, respectively. All blots were imaged using a ChemiDoc MP imaging system (Bio-Rad).
Additional methods are described in Supporting Information.

Results and Discussion
sgRNA(F+E) displays increased activity relative to the original sgRNA in deleting an MfeI restriction site in the klp-12 gene Initial reports describing use of oligos to introduce singlebase changes reported edits in 0.7-3.5% of the F 1 progeny screened . Recent work in mammalian cells reported that a modified sgRNA(F+E), with an extended Cas9 binding structure and removal of a potential PolIII terminator by an A-U basepair flip, exhibits improved activity (Chen et al. 2013a). I therefore tested whether this modified sgRNA(F+E) displayed increased activity in C. elegans, as a potential tool to improve editing efficiency. The modified sgRNA was introduced into the pDD162 CRISPR/Cas9 plasmid (Dickinson et al. 2013), and to evaluate sgRNA activity, deletion of an MfeI restriction site in the klp-12 gene ) was used as a readout ( Figure S1). sgRNA(F+E) produced a significant increase in deletion of the MfeI site in animals positive for the co-injection marker (Table 1), and was used for all subsequent experiments.
pha-1(ts) co-conversion increases knock-in efficiency with minimal handling Having increased sgRNA activity, I next turned to improving the screening process. Two recent reports describe that selection for a visible phenotype produced by a CRISPR/ Cas9 triggered editing event [i.e., unc-22 mutation or dpy-10(cn64) knock-in] results in an increase in knock-out and knock-in efficiencies at other genomic loci (Arribere et al. 2014;. These approaches can be used in any genetic background, but require outcrossing or meiotic segregation of the selective mutation. My aim was to develop a stringent system with minimal handling and no outcrossing or meiotic segregation of selection markers required. pha-1(e2123) is a temperature-sensitive embryonic lethal mutation; mutants are viable at 15°, but display embryonic and early larval lethality when cultivated at 25° (Schnabel et al. 1991;Granato et al. 1994). pha-1(e2123) has been previously used to select and propagate extrachromosomal arrays carrying a pha-1(+) marker (Granato et al. 1994). pha-1(ts) mutants display a low spontaneous reversion frequency of 2.5 3 10 25 per haploid genome (Schnabel et al. 1991); the stringency of this selection was confirmed by plating 144 gravid adults and shifting them to 25°; no viable progeny were produced, only dead eggs and arrested larvae. I selected a CRISPR/Cas9 target site that would produce a DSB 17 bp 39 from the e2123 point mutation and designed an 80mer repair oligo with the PAM silently mutated to prevent recleavage of the site in an edited animal ( Figure  1A). Twelve WT animals were injected with the CRISPR/ Cas9 plasmid, a co-injection marker, and a commercially purchased, PAGE purified repair 80mer. Eight rescued F 1 's were recovered, all heterozygotes for pha-1(ts) repair (data not shown). Although all eight F 1 's were positive for the coinjection marker, in later experiments, I also observed pha-1(ts) rescue in marker-negative animals. Thus, as previously observed (Arribere et al. 2014;), heritable transgenesis is not a prerequisite for efficient editing. Oligomediated repair of pha-1(ts) allowed recovery of repair heterozygotes, and the complete penetrance of the e2123 embryonic lethality meant that only F 1 heterozygotes (i.e., rescued animals) would develop, making screening extremely rapid.
A homozygote for pha-1(ts) repair had no significant difference in brood size compared to a WT control (Table S5; two-tailed t-test, P = 0.75).
I next tested whether pha-1(ts) repair could be used to enrich for knock-in of a 23FLAG oligo into the 39 end of the nhr-23 gene ( Figure 1B), a nuclear hormone receptor involved in molting and embryonic development (Kostrouchova et al. 1998(Kostrouchova et al. , 2001. I identified two potential PAMs near the desired insertion site that could be silently mutated ( Figure  1B). I designed a 200mer oligo to insert a 23FLAG epitope just before the stop codon. As half of sgRNAs have been reported to fail in C. elegans , I mutated two PAMs in the oligo to allow knock-in attempts with different sgRNAs. An 18-bp linker sequence encoding the flexible linker peptide glycine-serine-4xglycine (GSGGGG; Figure 1B) was included to spatially separate the 23FLAG tag from the NHR-23 C terminus, potentially facilitating accessibility of the tag for immunoprecipitation. Additionally, the tag encoded a BamHI restriction site, which was used for diagnostic restriction digestion in PCR-based screening ( Figure 1B). I injected a CRISPR/Cas9 plasmid targeting pha-1, an nhr-23 CRISPR/Cas9 construct targeting the PAM nearest the stop codon (PAM no. 1; Figure  1B), the 80mer pha-1(ts) repair oligo ( Figure 1A), and the 200mer nhr-23:23FLAG oligo ( Figure 1B); this oligo was not PAGE purified to test whether PAGE purification was a necessary cost. From 16 viable injected P0 animals, nine pha-1(ts) rescued F 1 progeny were obtained ( Figure 1D). These animals were plated, allowed to self-fertilize, and then single-worm genotyping was performed ( Figure 1C). Two of these nine animals were heterozygous for a potential 23FLAG insertion by diagnostic digest, of which one was a correct insertion; the other candidate had a frameshift in the 23FLAG epitope. F 2 progeny of the animal carrying the precise 23FLAG knock-in were singled and a homozygote for both the insertion and the repaired pha-1(ts) allele was obtained; both knock-ins were confirmed by sequencing. Broodsize analysis of a representative knock-in confirmed viability with no phenotype, indicative of nhr-23 loss of function (i.e., molting defects or high embryonic lethality) (Table S5).
To distinguish between pha-1(ts) repair homozygotes and heterozygotes without sequencing, I used CEL-1 digestion of PCR products ( Figure S2). CEL-1 is a celery endonuclease that recognizes and cleaves mismatches in double-stranded DNA (dsDNA) formed from single nucleotide polymorphisms or small insertions or deletions (Yang et al. 2000;Wood et al. 2011). In this assay, PCRs from repair heterozygotes will lead to a digestion product ( Figure S2). Normally, the absence of digestion could indicate that the animals were homozygous for either the ts allele or the repaired allele. However, growth at the restrictive temperature eliminates all ts homozygotes; therefore, all undigested PCR products are repair homozygotes. The genotypes predicted by CEL-1 digestion were confirmed by sequencing. CEL-1 or other mismatch cutting nucleases such as mung bean nuclease or T7E1 represent an efficient method to monitor single-basepair changes and help reduce the number of animals that need to be sequenced to identify homozygous knocked-in point mutations.

Short oligos are effective templates for gene conversion
The stringency of pha-1 co-conversion offered a powerful tool to optimize oligo editing parameters. As much experimental effort was spent isolating homozygotes to confirm correct epitope insertion, I designed primers that allowed sequencing of the 23FLAG tag in heterozygotes. These primers bound the insertion junction, with the two 39-most bases binding to the inserted sequence ( Figure S3). In some cases, poor sequencing quality made it difficult to confirm the sequence in the center of the epitope; in these cases, a separate PCR was performed using the epitope-specific primer and an external primer and sequencing was performed on this purified PCR product using the external primer. Screening in heterozygotes greatly reduced hands-on effort required to identify correct insertions. I next tested whether oligo length (200mer, 80mer, and 60mer) affected repair efficiency. The 80mer was PAGE purified, but the 200mer and 60mers were not. These oligos were all sense in relation to the pha-1 coding strand. The 80mer and 200mer produced similar numbers of pha-1 rescued F 1 's (9 and 12, respectively) and with the 80mer producing one nhr-23:23FLAG insertion and the 200mer producing four insertions ( Figure 1D). Interestingly, I observed a high percentage of males in the pha-1(ts) repaired F 1 , generated using the 200mer (Table S6). Despite the 60mer only producing three pha-1 rescues, two of those F 1 also had knock-ins at the nhr-23 locus. One knock-in was incomplete and produced a frameshift; the other had an extra insertion after the 23FLAG tag, but frame was maintained. I confirmed the expression of the 23FLAG tag by immunoblotting in five knock-in lines carrying a precise insertion of the 23FLAG tag, as well as a line that contained an imprecise insertion. All five precise knockins expressed the FLAG tag, whereas no band was seen for the strain carrying the frameshifted FLAG tag ( Figure  2). pha-1(ts) co-conversion allowed isolation of sequenceverified homozygotes for a knock-in event within 8-9 days.
Additional repair template considerations I then examined whether oligo polarity had an impact on editing, using a PAGE purified, antisense version of the 80mer pha-1(ts) repair oligo. The sense oligo was homologous to the coding DNA strand, whereas the antisense oligo was homologous to the template DNA strand. The antisense oligo produced the fewest knock-ins per viable P0 animal of all conditions tested, but still had a high rate of nhr-23::23FLAG knock-in per rescued F 1 ( Figure 1D). Thus, it appeared to be a poor repair template for pha-1(ts) repair, but once this repair event was selected could still lead to enrichment in nhr-23::23FLAG knock-in. To further explore this variable, I tested whether nhr-23::23FLAG could be knocked in using an antisense 200mer. Using a sense pha-1 200mer, five rescued F 1 's were obtained from 57 viable P0 animals, but no nhr-23::23FLAG knock-ins were detected ( Figure 1D). Including the antisense 200mer with the sense 200mer did result in a decrease in knock-in efficiency (1/5 vs. 1/9), but more animals need to be screened to determine if this effect is significant ( Figure   S4). Interestingly, when annealed nhr-23::23FLAG sense and antisense oligos were injected, no knock-ins were recovered, indicating that dsDNA is not a better template than single-stranded DNA (ssDNA) and may be less effective ( Figure  S4). These data suggest that the strand to which oligo homology is derived could be an important parameter for editing efficiency and that ssDNA may be more effective than dsDNA for epitope knock-in. However, these inferences must be tested with many additional combinations of sgRNAs, loci, and repair templates to assess their generality.
Growth on HB101 suppresses pha-1(ts) low brood size and sterility A number of injected P0s in these experiments were sterile. This sterility could have been a consequence of injection trauma or general sickness of pha-1(ts) mutants. To discriminate Figure 1 Selection for pha-1(ts) oligo-mediated repair enriches for nhr-23::23FLAG knock-in. (A) Sequence of the pha-1(e2123) genomic locus targeted, with the PAM (red text), e2123 G-to-A mutation (boldface text and underline), sgRNA target sequence, and position of the DSB indicated. The 80mer repair oligo also contains a silent C-to-A mutation to inactivate the PAM. (B) Sequence of the nhr-23 genomic locus targeted. The stop codon (blue text), PAM (no. 1, red text) and sgRNA target, the DSB position, and an alternate PAM (no. 2, red text) are indicated. The 200mer repair oligo was designed to insert a 23FLAG epitope with a flexible GSGGGG linker sequence, which also contains a BamHI site. The oligo contains silent C-to-A and G-to-A mutations to inactivate the two indicated PAMs. (C) (i) pha-1(ts) mutants propagated at the permissive temperature were injected with 60 ng/ml each of CRISPR/Cas9 plasmids targeting the PAM in pha-1(ts) and PAM no. 1 in nhr-23, 50 ng/ml of an oligo designed to correct the pha-1(ts) allele, and 50 ng/ml of the 200mer nhr-23::23FLAG repair oligo. (ii) P0 animals were singled onto individual plates and shifted to the restrictive temperature (25°). (iii) Three to four days later, the plates were screened for the presence of viable progeny (L3s to adults); the e2123 embryonic lethality phenotype is completely penetrant at 25°, and only rescued animals develop. Rescued F 1 progeny were singled onto individual plates, allowed to lay eggs (2-3 days), and the parental F 1 was genotyped by PCR followed by BamHI digestion. Correct insertion of the epitope is confirmed by sequencing-purified PCR products with knock-in-specific primers. (iv) Homozygotes were recovered by plating 12-24 progeny from candidate nhr-23:23FLAG knock-in F 1 's, allowing them to lay eggs (2-3 days), and genotyping the parental F 2 animal by PCR and BamHI digestion. Marker size in kilobases is provided. (D) Summary of pha-1(ts) co-conversion experiments. Viable P0 are the number of injected animals that produced eggs; a variable number of animals are sterile in each experiment. The length and polarity (with respect to the coding strand) of the pha-1(ts) repair and nhr-23::23FLAG oligos are provided. P0 were propagated on OP50 E. coli for these injections.
between these possibilities, I shifted adult pha-1(ts) mutants to 25°to mimic the selection protocol. Interestingly, these animals displayed sterility (1/24 P0) and low brood sizes (,10 eggs) in 6 of 24 P0s. Growth on HB101 has been shown to suppress the slow growth rate of eat-2 (ad465) and eat-5 (ad1402) mutants, which have defective pharyngeal pumping (Shtonda and Avery 2006). Moreover, unc-119(ed3) strains are frequently grown on HB101 in genome editing protocols to ameliorate sickness of that strain (Frøkjaer-Jensen et al. 2010;Dickinson et al. 2013). I therefore tested the effect of HB101 growth on pha-1(ts) mutants by propagating animals for several generations on HB101 at 15°, then picking adult animals and shifting them to 25°. Growth on HB101 suppressed the low brood size and sterility observed during growth on OP50. pha-1(ts) mutants grown on HB101 and then shifted to OP50 at L4 had brood sizes of 127 6 34 (Table S5) with 0% viable progeny. Subsequently, the pha-1(ts) strain was maintained on HB101 at 15°prior to micro-injection.

A PCR fusion method to rapidly generate new sgRNA templates
Having demonstrated that oligo-mediated repair of the pha-1(e2123) mutation can be used to efficiently enrich for knock-ins at other loci, I next turned to optimizing sgRNA delivery. Generation of new sgRNA templates for plasmidbased CRISPR/Cas9 systems typically involves site-directed mutagenesis (Dickinson et al. 2013) or cloning using oligonucleotides (Waaijers et al. 2013) or PCR products . A major impediment to the success of a CRISPR/Cas9 experiment is the efficiency of the sgRNA;  report that half of their tested sgRNAs fail. Given that efficient promoter::GFP reporters can be generated by fusion PCR (Hobert 2002), I tested whether the same approach could be used to express sgRNAs from the U6 promoter. This approach would allow rapid screening of sgRNAs, as PCR templates could be injected on the same day of amplification without need for cloning or transformation. For cost efficiency, four PCR primers were used, of which only one is changed to generate a new PU6::sgRNA template ( Figure 3A). As a proof of principle, I generated PU6::sgRNA template fusions targeting pha-1 and nhr-23 PAM no. 1, and deleted the sgRNA template sequence from the pJW1219 Cas9/CRISPR plasmid. Injecting this Cas9 plasmid (pJW1259), the PCR-generated pha-1 and nhr-23 PU6::sgRNA templates, the pha-1(ts) sense 80mer repair template, and the nhr-23:23FLAG sense 200mer, efficient knock-in at both loci with comparable frequency to the corresponding plasmid-based PU6::sgRNA templates was observed ( Figure 3B). Fusion PCR thus allows rapid, cost-effective generation of sgRNA templates driven by the U6 promoter.
DSBs up to 54 bp from the insertion site can generate nhr-23::23FLAG knock-ins During my initial design of the nhr-23::23FLAG repair oligo, I mutated two potential PAMs, as it has been reported that half of all sgRNAs fail; this single oligo would allow testing of two separate sgRNAs . While analyzing the sequence of the 22 candidate nhr-23::23FLAG knock-ins generated using sgRNAs targeting PAM no.1 and identified by BamHI digest ( Figure 1D, Figure 3B, Table 2), I observed co-conversion of the PAM no. 2 silent mutation in six lines. Figure 2 Detection of NHR-23::23FLAG in precise knock-ins. Four micrograms of protein from synchronized gravid adults of the indicated strains was analyzed by immunoblotting with anti-FLAG. KRY48 contains a frameshift in the 23FLAG tag and thus does not express the epitope. Stain-free (Bio-Rad) analysis of total protein on the blot is provided as a loading control. Marker size (in kilodaltons) is provided.
These co-conversion events suggested that pha-1(ts) selection could be used to explore gene conversion track lengths and effectiveness of DSB position relative to the insertion site.
The silent point mutations in PAMs no. 1 and no. 2 were 29 bp apart, which agrees with my observation that a 60mer with 29 bp of homology can be used to efficiently knock-in point mutations (Figures 1D and Figure 4A). For these next experiments, I sequenced all pha-1(ts) rescued F 1 's. I first tested whether an sgRNA targeting PAM no. 2 could be used to knock in the 23FLAG tag into the nhr-23 39 end; 3 of 12 pha-1(ts)-rescued F 1 animals had the PAM no. 2 mutation knocked in, with one animal also containing the PAM no. 1 mutation and correct insertion of the 23FLAG tag ( Figure  4B, Table S7). I next examined whether a DSB 54 bp away from the desired insertion site could be used to knock in the 23FLAG epitope ( Figure 4A). I designed a modified version of the 200mer used for the above experiment that also carried three silent mutations to disrupt the sgRNA binding to the PAM no. 3 target site, as well as the mutations in PAMs no. 1 and no. 2 ( Figure 4A). A fourth potential PAM (no. 4) was left intact in the repair template, as inactivation would leave only 10 bp of 59 homology; this sgRNA was inactive (Figure 4, A and B). Using an sgRNA targeting PAM no. 3 to generate a DSB, two of eight pha-1(ts)-rescued F 1 's carried the silent mutation in PAM no. 3; the others all had wildtype sequence, suggesting that this may be an inefficient sgRNA ( Figure 4B, Table S7). Of the two animals with mutations in PAM no. 3, both had the PAM no. 2 inactivating mutation, and one had the PAM no. 1 mutation and insertion of the 23FLAG tag (Table S7). Though there was a 1-bp deletion within the tag, this experiment demonstrates that a DSB 54 bp from an insertion site can be used for introduction of epitopes, and that the presence of mutations in a stretch of homology does not prevent gene conversion; five point mutations in a 55-bp stretch were introduced into the genome along with the 65 bp of 23FLAG tag (with the 1-bp deletion). Animals were not tracked to identify originating P0s; however, for the PAM no. 1 knock-in experiment, the six animals with PAM no. 2 mutation co-conversion were isolated from six different injections, and thus represent independent events ( Figure 4B). Knock-in efficiency appeared higher the closer the DSB was to the insertion site, but sgRNA efficiency could also play a role in this observation.
Homology arms of 35 bp are sufficient for oligo-templated insertion of a 23FLAG tag Exploring DSB position relative to the insertion site demonstrated that the stringency of pha-1(ts) co-conversion combined with the speed and ease in recovering editing events allowed rapid testing of editing parameters. Interestingly, the 200mer used for the PAM no. 3 knock-in experiments only had 29 bp of homology 59 to the DSB (Figure 4, A and  B), suggesting that homology arms could be relatively short. Efficient repair of the pha-1(ts) allele was observed using a 60mer with 29 bp of homology ( Figure 1D). Furthermore, recent data using PCR-derived dsDNA repair templates demonstrated Figure 3 PCR generated sgRNA templates can be used for pha-1(ts) co-conversion. (A) Method for generating PU6::sgRNA templates by PCR. The U6 promoter and sgRNA template were separately subcloned to generate pJW1310 and 1311, respectively. The U6 promoter is amplified with oligos 1 and 2, and the sgRNA template is amplified with oligos 3 and 4. Oligo 3 contains 20 bp of homology to the sgRNA template, the new 20 bp sgRNA targeting sequence, and 20 bp of homology to the U6 promoter. The resulting PU6 and sgRNA template PCR products are then mixed and used as template in a second PCR reaction using oligos 1 + 4. (B) Animals were injected with 60 ng/ml of the Cas9 plasmid, 25 ng/ml each of PCR generated PU6::sgRNA templates targeting pha-1 and nhr-23 (same targeting sequence as in Figure 2), and 50 ng/ml each of the 200mer sense pha-1(ts) repair oligo and nhr-23::23FLAG oligo. Viable P0 are the number of injected animals that produced eggs; a variable number of animals are sterile in each experiment. P0 were propagated on HB101 E. coli for these injections. Oligo polarity (sense) is with respect to the coding strand.
that 30-to 60-bp homology arms were optimal, with knock-in efficiency actually decreasing with longer homology arms (Paix et al. 2014). To test the ideal length of homology for oligomediated insertion of epitopes, I designed nhr-23::23FLAG oligos with 35 bp, 25 bp, or 15 bp of homology and tested their ability to introduce the 23FLAG epitope using a DSB generated by an sgRNA targeting PAM no. 1. Knock-in efficiency using these homology arms was compared to pooled data from the pha-1(ts) and nhr-23::23FLAG sense oligo experiments ( Figure  1D and Figure 3B). This 200mer sense nhr-23::23FLAG oligo ( Figure 1B) contained 54 bp of homology 39 to the insertion site and 76 bp of homology 59 to PAM no. 1 (76/54). With the 35-bp homology arms, two animals from 9 rescued F 1 's had correct insertion of the 23FLAG epitope ( Figure 4C). This 22% knock-in per F 1 screened (2 of 9) is comparable to the 26% efficiency (7 of 27) observed using the 76/54 oligo ( Figure 4C). The knock-in rate per successfully injected P0 was also comparable for the 35-bp and the 76/54-bp homology arms [4.76% (2/42) vs. 6.86% (7/102)]. The 15-and 25-bp homology arm produced fewer pha-1(ts) rescues per successfully injected P0, and only the 25-bp homology arms could produce a 23FLAG insertion, though this knock-in carried a point mutation ( Figure 4C). These data suggest that, similar to dsDNA templates, homology arms of 35-80 bp are ideal for oligo-templated editing.

Inactivation of NHEJ repair increases knock-in efficiency
In Drosophila, increased homologous recombination efficiency can come from inactivation of NHEJ (Beumer et al. 2008;Bottcher et al. 2014). I therefore used the pha-1(ts) system to test whether NHEJ inactivation impacted knock-in by homologous recombination. NHEJ mutants may have additional background mutations due to compromised repair of endogenous DSBs and would require additional outcrossing. I therefore tested the effect on knock-in frequency of temporary inactivation of the C. elegans homolog of Ku80 (cku-80), part of a heterodimer that binds the end of DSBs in NHEJ, and which had reported RNAi phenotypes (Dmitrieva et al. 2005;McColl et al. 2005). pha-1 and nhr-23 CRISPR/Cas9 plasmids, the pha-1 sense 80mer repair oligo, and the nhr-23 sense 200mer repair oligo were injected into pha-1(ts) mutants treated with control or cku-80 RNAi. Control RNAi produced one pha-1(ts) rescued F 1 , which also carried a sequence-confirmed nhr-23 knock-in event (Table 2). However, inactivation of cku-80 by RNAi produced an increase in both pha-1(ts) rescue (10 F 1 's) and nhr-23:23FLAG co-knock-ins recovered (n = 5) ( Table 2). These experiments suggested that NHEJ inactivation boosts oligo-mediated knock-in efficiency.
To confirm that pha-1(ts) co-conversion, PCR-generated sgRNAs templates, and NHEJ inactivation were effective on other loci, I attempted to introduce a 23FLAG epitope in nhr-25, a broadly expressed nuclear hormone receptor that regulates several developmental and physiological programs (Asahina et al. 2000;Gissendanner and Sluder 2000;Chen et al. 2004;Asahina et al. 2006;Mullaney et al. 2010;Ward et al. 2013Ward et al. , 2014. I selected the 39 end of nhr-25 as there are two isoforms that share a common 39 end, but differ in promoter use; thus, targeting the 39 end of nhr-25 with the 23FLAG should in principle allow for labeling of all known nhr-25 isoforms. I designed a 175mer repair template with six silent point mutations in the 20-bp sequence preceding the PAM, as the PAM could not be silently mutated ( Figure  S5, A and B). As with the nhr-23::23FLAG construct, an 18-bp spacer encoding with a BamHI site was also included ( Figure S5, A and B). This oligo contained 41 bp of homology 59 to the mutated sgRNA target sequence and 43 bp of 39 homologous sequence. pha-1(ts) mutant animals grown on either control or cku-80 RNAi were injected with a pha-1(ts) CRISPR/Cas9 plasmid, an nhr-25 PU6::sgRNA template PCR product, and pha-1(ts) and nhr-25::23FLAG repair oligos. Seven pha-1 rescues were recovered from the control RNAi-treated animals and 36 from the cku-80 RNAi-treated animals ( Table 2). No knock-in candidates were recovered from the control RNAi injection, but five were recovered pha-1(ts); control(RNAi) a 80mer nhr-23:::23FLAG from the cku-80 RNAi-treated animals, of which four were correct insertions of the 23FLAG tag (Table 2). Strikingly, one of these animals was an F 1 homozygous knock-in. These strains were viable and did not display defects associated with nhr-25 inactivation (protruding vulvae, molting defects, embryonic lethality; see Table S5 for a representative brood size). I confirmed expression of the 23FLAG tag in an outcrossed, representative line ( Figure 5B).

Multiplexed editing using pha-1(ts) co-conversion and cku-80 RNAi
Given the efficient editing observed in cku-80 RNAi-treated animals and the 35-80 bp of homology sufficient for oligo-templated repair, I next tested whether I could introduce larger epitopes using pha-1(ts) co-conversion. As  had reported generating mutations in both avr-14 and avr-15 in one unc-22 co-CRISPR experiment, I attempted to knock in 84-bp 3xFLAG tags containing the GSGGGG spacer into both nhr-23 and nhr-25 in one injection experiment ( Figure S5A). From 13 successfully injected P0 animals treated with cku-80 RNAi, two precise nhr-23::3xFLAG lines were obtained (Table 2). A viable nhr-25::3xFLAG line that expressed the 3xFLAG tag, but had a 51-bp duplication in the 39 UTR was also obtained; as the tag was expressed and the strain was viable, this line was scored as a correct knock-in (Table 2, Table S5). A single knock-in animal was  Table 2); only animals that displayed a knock-in signature in the diagnostic BamHI digest from these experiments were sequenced. For the PAM no. 2, no. 3, and no. 4 experiments, all pha-1(ts) F 1 rescued animals were sequenced. (C) Testing homology arm length on nhr-23::23FLAG knock-in efficiency. Homology lengths in basepairs for 59 and 39 arms are provided. 59 arm homology numbering starts from the middle G in PAM no. 1; 39 homology numbering starts from the first basepair of the stop codon. Animals were co-injected with 50 ng/ml each of pha-1 targeting and nhr-23 PAM no. 1 targeting CRISPR/Cas9 plasmids, 50 ng/ml each of the pha-1(ts) repair oligo and nhr-23::23FLAG repair oligo. For the 76/54-bp homology arm row in the table, data were pooled from all non-RNAi experiments using pha-1 sense repair oligos and nhr-23::23FLAG sense 200mers (Figure 1 and Figure 3). With the exception of the pooled data, all injected P0 animals were grown on HB101 E. coli in B and C. Oligo polarity (sense) is with respect to the coding strand.
obtained from 34 successfully injected P0 animals grown on control RNAi (Table 2). This F 1 animal was homozygous for an nhr-25::3xFLAG insertion and heterozygous for an nhr-23::3xFLAG insertion (Table 2). Viability was confirmed for representative nhr-23::3xFLAG and nhr-25::3xFLAG lines and for the nhr-23::3xFLAG; nhr-25::3xFLAG double knock-in line (Table S5). No defects consistent with nhr-23 or nhr-25 inactivation were observed, though the double knock-in did have a lower brood size (Table S5). I confirmed FLAG epitope expression in outcrossed nhr-23::3xFLAG lines and nhr-25::3xFLAG lines ( Figure 5, A and B). The 3xFLAG epitope lines displayed a marked increase in band intensity in immunoblots compared to 23FLAG lines, for both NHR-23 and NHR-25 ( Figure 5, A and B). Notably, the nhr-23::23FLAG epitope was not detectable in this experiment ( Figure 5A), as a more potent ECL substrate was required for detection by immunoblotting ( Figure 2); this more potent ECL substrate may explain the nonspecific band observed in Figure 2, but not in Figure 5A. I also observed extra bands in the 3xFLAG lines for NHR-23 and NHR-25 compared to the corresponding 23FLAG lines, which would be consistent with NHR-23 and NHR-25 isoforms, though they could also represent degradation products ( Figure 5, A and B). Comparing all NHEJ inactivation experiments, growing P0 animals on cku-80 RNAi produced a significant increase in the number of knock-ins recovered per injection experiment (two tailed t-test P = 0.01) and percentage of knock-ins per viable P0 (two-tailed t-test P = 0.016). No significant difference was observed in the number of P0 animals with rescued F 1 progeny (two tailed t-test P = 0.16) or in the percentage of knock-ins per rescued F 1 (two tailed t-test P = 0.85). Recent manuscripts have described a "jackpot" phenomenon (Arribere et al. 2014;Paix et al. 2014), where the majority of edits come from a small number of P0 animals. There may be a trend of "richer jackpots" in cku-80 RNAi-treated animals with 2.6 rescued F 1 's/P0 producing F 1 rescues (60.44; 95% confidence interval) vs. 1.6 (60.50; 95% confidence interval) in control RNAi-treated animals. This difference was not significant in a two-tailed t-test (P = 0.19) potentially due to the large variation in number of F 1 rescues and knock-ins produced across assays, and this observation will require further exploration.

PAGE purification of oligos is not necessary, but increases editing efficiency
The nhr-25::3xFLAG oligo appeared to be a poor repair template, as it yielded lower knock-in efficiency in comparison to the nhr-25::23FLAG template, and complex insertions were observed in two nhr-25::3xFLAG candidate knock-in lines.
Although experiments demonstrated that PAGE purification was not necessary, the nhr-25::3xFLAG template was ideal to test whether PAGE purification nonetheless increased knock-in efficiency or fidelity. Resolving unpurified and purified oligos on a denaturing TBE-Urea gel revealed that the unpurified oligo preparation contained large amounts of incorrectly sized product ( Figure S6A). pha-1(ts) animals grown on control or cku-80 RNAi were injected with the pha-1 targeting CRISPR/ Cas9 vector, an nhr-25 sgRNA template PCR product, a pha-1 repair oligo, and either purified or unpurified nhr-25::3xFLAG 175mers. Interestingly, this experiment demonstrated that PAGE purification resulted in higher rates of knock-ins per F 1 and per viable P0 ( Figure S6B). Comparing all experiments using the nhr-25::3xFLAG oligos (Table 2, Figure S6C), both knock-in candidates obtained using the purified oligo were precise insertions, whereas two of the four candidates generated with the unpurified oligo contained additional sequence inserted ( Figure S6C). These experiments suggested that one can either opt for an increased knock-in rate with PAGE purified oligos or avoid this cost and inject/screen more animals.
Efficient editing at other genomic loci and rapid testing of sgRNA activity using pha-1(ts) selection and NHEJ inactivation Finally, I wished to test these editing parameters on other genes to test their general applicability and robustness. Based on my previous work on NHR-25 sumoylation , I chose a previously described weakly efficient sgRNA ) that cleaved 19 bp 39 to the start codon of the single C. elegans SUMO gene, smo-1 (Broday et al. 2004). I designed a 175mer repair oligo to create a SMO-1 N-terminal 23FLAG fusion ( Figure S5, C and D). I also designed two overlapping sgRNAs to target the NHEJ gene, lig-4, and a 60mer oligo as the homologous recombination template to insert a stop codon (oligos were unpurified). From nine successfully injected P0 animals grown on cku-80 RNAi and injected with PCR-derived sgRNA templates, 27 pha-1(ts) rescued F 1 animals were obtained. Fourteen of these F 1 's carried knock-ins in smo-1, of which 11 were precise (Table 2). I confirmed the expression of the 23FLAG epitope in a representative line (KRY82) by immunoblotting; signal was observed over a large range of molecular weights, as expected from SUMO conjugation to hundreds of substrates in C. elegans (Kaminsky et al. 2009) ( Figure 5C). Although viable, 23FLAG::smo-1 homozygotes displayed partially penetrant phenotypes consistent with smo-1 reduction of function, such as small body size and protruding vulvae. Thus, epitope placement may need to be optimized in smo-1, though the creation of a viable hypomorph will be a useful reagent for the community. The lig-4 sgRNAs failed, as no knock-ins or mutations were obtained in lig-4, demonstrating that this approach also allows rapid testing of sgRNA efficacy, similar to   (Table  2). Together, these results demonstrate that a co-conversion approach using a temperature-sensitive mutant allele, PCR-generated sgRNA templates, and NHEJ inactivation by RNAi provide a flexible, robust platform to recover genome editing events.

pha-1(ts) coselection increases screening efficiency
Based on recent co-CRIPSR/co-conversion reports (Arribere et al. 2014;, and the frequency at which relatively rare pha-1(ts) repair events were associated with edits at other loci, it was highly probable that pha-1(ts) coselection increases screening efficiency. Prior to developing pha-1(ts) co-conversion, I had attempted to introduce 23FLAG tags onto the 39 end of nhr-23 and nhr-25 and recover knock-ins through direct screening of F 1 progeny, similar to the approach described by Paix et al. (2014) (see Supporting Information for detailed methods). No knock-ins were recovered from 380 screened WT F 1 's (Table S8). I also tested the effect of NHEJ inactivation by injecting into lig-4(ok716) mutants; DNA ligase 4 (lig-4) encodes the enzyme that seals DSBs in canonical NHEJ, and ok716 is an out-of-frame deletion that removes the catalytic ligase domain and is predicted to result in a premature stop codon (Clejan et al. 2006). A single knock-in was recovered from 768 screened lig-4 mutant F 1 's (Table S8); after outcrossing the lig-4 mutation, this nhr-25::23FLAG line had a brood size equivalent to a knock-in produced by pha-1(ts) coselection (Table S5). In comparable pha-1(ts) coselection experiments, no knock-ins were recovered from 7 F 1 laid by control RNAi treated P0s and four knock-ins were recovered from 36 F 1 's laid by cku-80 RNAitreated P0 (Table 2). For nhr-23::23FLAG knock-in experiments, I used a knock-in-specific PCR screening approach; as in optimization experiments, it detected knock-in DNA diluted 1:1280 with WT DNA, whereas diagnostic restriction digestion of PCR products could only detect up to a 1:20 dilution of knock-in DNA with WT DNA ( Figure S7). No knock-ins were recovered from 200 WT F 1 animals or 840 lig-4 mutant F 1 's (Table S8). In comparison, pha-1(ts) coselection using a similar sense nhr-23::23FLAG repair template yielded seven knock-ins from 27 screened pha-1(ts) rescued F 1 's ( Figure 4C). Comparing all similar nhr-23::23FLAG and nhr-25::23FLAG direct selection and pha-1 co-conversion experiments (i.e., excluding antisense oligo and NHEJ inactivation experiments) demonstrated a significant increase in screening efficiency compared to direct F 1 screening (twotailed t-test, P = 0.01).

Conclusions
Here, I demonstrate that selection for repair of a temperaturesensitive point mutation using an oligonucleotide template can be used to efficiently select for knock-in at other loci in as quickly as 8-9 days. Several findings of this work should be applicable for a range of co-conversion/co-CRISPR approaches.
First, the increased sgRNA(F+E) activity (Table 1) may improve knock-in and knock-out efficiencies and reduce the number of sgRNAs that one must test to identify an active sgRNA. Second, for introduction of single-basepair changes, efficient editing was observed using 60mers, 80mers, and 200mers. There may be a slight improvement in knock-in efficiency with longer repair templates, but using shorter oligos still produces repair events at a high rate. These data are consistent with the 29-bp repair track length observed using the second silent PAM mutation in the nhr-23::23FLAG repair oligo and the efficient nhr-23::23FLAG knock-ins obtained using 35 bp of homology (Figure 4). This optimal homology for oligo-mediated editing is similar to that reported for dsDNA templates (Paix et al. 2014). The 84-bp 3xFLAG nhr-23 and nhr-25 knock-ins is a larger insertion than described by several recent reports (Lo et al. 2013; Figure 5 Detection of FLAG epitope expression in nhr-23-, nhr-25-, and smo-1tagged lines. Anti-FLAG immunoblot analyses of lysates are from mixed stage animals of the indicated genotypes. The 2x and 3xFLAG tagged nhr-23 (A) and nhr-25 (B) lines and a 23FLAG::smo-1 tagged line (C) were assayed. Stain-free (Bio-Rad) analysis of total protein on each blot is provided as a loading control. Marker size (in kilodaltons) is provided. The same exposure time was used to image all anti-FLAG blots. For the NHR-23 blot (A), the background band observed in Figure 2 was not detected, likely because a more potent ECL substrate was used for that experiment. Arribere et al. 2014;Paix et al. 2014;); a 66-bp 3xFLAG insertion in the nos-1 gene is the most comparable edit (Paix et al. 2014). Based on the minimal homology length of 35 bp and current oligo synthesis limit of 200 bp, it may be possible to knock in sequences of up to 130 bp using oligo templates. Third, I demonstrated that oligos do not need to be PAGE purified, as efficient editing was observed using unpurified 60mers and 200mers to edit the pha-1(ts) allele and to introduce the nhr-23::23FLAG epitope ( Figure 1D). However, PAGE purified oligos did result in increased knockin efficiency and fewer imprecise knock-ins ( Figure S6). Thus, an investigator can choose between the additional cost for the increased editing efficiency or simply inject more P0 animals and screen more F 1 's. It is very important to note that there is likely wide variation in quality of unpurified oligos between different suppliers. Different preparations may contain different inhibitors or cytotoxic compounds and one should confirm the efficiency of unpurified oligos from a new supplier by testing a control such as pha-1(ts) rescue or dpy-10(gf) knockin (Arribere et al. 2014). Fourth, PCR-generated PU6::sgRNA templates allow rapid production of new sgRNAs without need for cloning. Moreover, the robust sgRNA activity observed when injecting linear dsDNA templates enables other technologies for sgRNA template production such as gene synthesis or oligonucleotide arrays (Bassik et al. 2009;Gilbert et al. 2014), which could be used to create pooled sgRNA template libraries. Injecting multiple PCR-generated PU6::sgRNA templates allows several editing experiments to be performed simultaneously. From single-injection experiments, I was able to knock 3xFLAG tags into both the nhr-23 and nhr-25 loci or generate 23FLAG:: smo-1 lines while also determining that the lig-4 sgRNAs were inactive (Table 2). Finally, the observation that inactivation of NHEJ can lead to improved knock-in rates can be adapted for any HR-based editing system, though it would reduce the efficiency of systems that rely on coselection of CRISPR-generated mutations as a marker. The increased knock-in efficiency observed following NHEJ inactivation was surprising and suggests that at least some knock-ins must be occurring outside in the germline; previous work has shown that NHEJ is actively suppressed in meiosis to ensure faithful repair of DSBs by homologous recombination (Adamo et al. 2010). Determining the cell types in which knock-ins are occurring and the DNA repair pathways involved could lead to improvements in editing efficiency.
It was intriguing that, for pha-1 and nhr-23, higher editing efficiency was observed using oligos with homology to the coding strand. In these experiments, the sgRNA for pha-1 recognized the coding strand, while the nhr-23 PAM no. 1 sgRNA recognized the template strand ( Figure 1D, Table S9). Although numerous explanations could be invoked for a polarity bias (sgRNA sequestration of the oligo, oligo:mRNA hybridization, different secondary structures in a given oligo and its reverse complement, etc.), it is unclear whether my data reflect a biologically meaningful trend or a chance observation. Best practice should be to clearly report the strand to which the sgRNA binds and the strand to which the oligo contains homologous sequence (Table S9). A metaanalysis of many more combinations of sgRNAs and oligos of differing polarities at a large number of genes is required to definitively determine whether oligo polarity is an important parameter. If one fails to obtain oligo-templated knock-ins when using a sgRNA with confirmed activity, then it may be worth testing the complement of the oligo.
The pha-1(ts) approach is distinct from reported co-CRISPR and co-conversion methods (Arribere et al. 2014; in that it starts with a mutant animal and results in restoration of a wild-type animal. In contrast, the other two approaches can be used with any strain, but require outcrossing or meiotic segregation of the selection marker. pha-1(ts) co-conversion may be advantageous as a marker in cases where the desired insertion site is linked to the selectable marker site. Arribere et al. (2014) demonstrated iterative editing using dpy-10(gf) co-conversion. pha-1(ts) co-conversion should allow for iterative editing events, though not as elegantly as the Arribere approach. pha-1(ts) F 1 rescues have all been heterozygous for the repair event. By shifting to the permissive temperature (15°) after generating homozygous animals of a desired knock-in, one could reisolate the pha-1(ts) allele and perform another round of editing; this pha-1(ts) reisolation would require an additional 5 days. Alternatively, one could simply cross the pha-1(ts) allele back into an edited strain.
The optimizations I report make oligo-mediated editing efficient, cost effective, and can be applicable to any CRISPRmediated editing system. The development of three distinct variations of coediting selection (unc-22 mutation; , rol-6(gf)/sqt-1(gf)/dpy-10(gf) knock-in (Arribere et al. 2014), and pha-1(ts) repair (this article) highlight the robustness of this method to select for genome editing events. The recent description of PCR-derived dsDNA templates (Paix et al. 2014) will make these approaches even more powerful; the abundance of potential genetic markers in other model organisms should make these widely applicable approaches. A current challenge in cell-culture-based systems has been the laborious recovery of rare knock-in events. Coselection markers such as oligo-mediated repair of mutated GFP or drug resistance cassettes, or inactivation of hypoxanthine phosphoribosyl transferase, could yield similar improvements in editing event recovery in these systems.

Acknowledgments
I thank Teresita Bernal, Soledad de Guzman, and Greg Wright for experimental assistance; Dan Dickinson, Te-Wen Lo, Barbara Meyer, Rob Nakamura, Axel Bethke, and members of the Yamamoto lab for helpful discussions and technical advice; Lindsey Pack, Gabriela Monsalve, Debbie Thurtle, Elizabeth Silva, Stefan Taubert, Kaveh Ashrafi, and Keith Yamamoto for advice and comments on the manuscript; and the reviewers for their many excellent comments and experimental suggestions. Some strains were provided by the CGC, which is funded by National Institutes of Health (NIH) Office of Research Infrastructure Programs (P40 OD010440). J.D.W. was supported by the National Institute Of General Medical Sciences of the NIH under award no. K99GM107345. Additional support was from NIH (CA20535) and U.S. National Science Foundation (MCB 1157767) awards to K. Yamamoto. The content is solely the responsibility of the author and does not necessarily represent the official views of the NIH.  indicated. The oligo is designed to bind the genomic sequence at the insertion site with the last two bases binding bases in the insert. In cases where sequence is too poor to confirm correct insertion of an epitope, an additional round of PCR can be performed using one of the insert-specific oligos and an external primer that binds in the genomic sequence. Purification of this product followed by sequencing using the primer that binds in the genomic sequence provides the entire epitope sequence. In events where 2 bp of knock-in sequence is not sufficient to confer specificity, increasing the knock-in specific homology will correct the problem at the expense of sequencing coverage of the knock-in. Figure S4 dsDNA is not a more effective template than ssDNA for introduction of a 2xFLAG epitope at the 3' end of nhr-23. (A) 50 ng/µl of sense of and antisense nhr-23::2xFLAG oligos in annealing buffer (TE buffer with 50 mM NaCl) were either annealed by heating to 95ºC for two minutes and then slowly cooling to 25ºC over 30 minutes in a thermocycler, or mock annealed (kept at 25ºC). Annealing was confirmed by resolving the annealed and mock annealed oligos on a 4% TAE-agarose gel and staining with GelRed. The 1KB+ (Invitrogen) size standard is provided. (B) Table comparing the knock-in efficiencies of sense oligos, and either mock annealed or annealed sense+antisense nhr-23::2xFLAG 200mers. The sense 200mer data is pooled from all experiments using pha-1(ts) sense oligos and nhr-23::2xFLAG sense 200mers (Figures 1 and 3). A control where the sense oligo was injected in annealing buffer was performed to ensure that the buffer did not affect knock-in efficiency.

Figure S7 Detection of knock-ins by knock-in specific PCR and diagnostic restriction digestion. (A) For
nhr::23::2xFLAG direct screening, a knock-in specific PCR approach was developed to minimize the number of PCRs required to identify knock-ins. Using the nhr-25::2xFLAG strain generated by direct selection (Table S8) as a control, oligos were designed to bind within the inserted sequence (oligo #1715) and outside of the insertion area. nhr-25::2xFLAG knock-in lysate was diluted as indicated with WT lysate and used as template in a knock-in specific genotyping PCR. No product was detected in the absence of nhr-25::2xFLAG lysate and knock-in product could be detected across the dilution range, to 1:1280. (NTC; no template control). (B) Identification of nhr-25:2xFLAG knock-in by diagnostic restriction digest. nhr-25::2xFLAG lysate was diluted and used in genotyping PCR as in (A). The product was then digested by BamHI to detect knock-ins. Knock-in product could be detected up to a 1:20 dilution. The 1KB+ (Invitrogen) size standard is provided in A and B.

PCR-based knock-in screening
For direct screening of nhr-25::2xFLAG knock-ins, WT animals or lig-4(ok716) mutants were injected with 100 ng/µl of pJW1185 (nhr-25 targeting CRISPR/Cas9), 10 ng/µl of a myo-2::tdTomato co-injection marker, and 100 ng/µl of a nhr-25::2xFLAG 135 mer (oligo #1580, Table S2). P0 animals were plated in single wells of 12-well plates containing NGM-lite agar seeded with OP50 E. coli. Following incubation at 25ºC for three days, wells were scored for the presence of marker positive F1 progeny. From these wells, the marker positive F1 were picked off and discarded and 1 ml of M9+gelatin was added. This step was performed because previous reports suggested that edits occurred in marker negative F1 . Marker negative F1s from these wells were pipetted into 30 µl of M9+gelatin in a 96-well plate; four worms were pipetted into each well. A multichannel pipette was used to add 30 µl of 2xOP50 food to each well. This food was made by inoculating a one liter culture of LB+streptomycin (50 µg/ml) with a single colony of OP50, shaking for 16 hours at 225 rpm at 37ºC, pelleting the culture by spinning at 4000 rpm, and resuspending in 10 ml of M9. For the 2xOP50 food, 5 ml of this concentrated OP50 was added to 45 ml of M9+gelatin containing 10 µg/ml cholesterol. Plates were parafilmed and incubated at 25ºC for 3-4 days. Lysates were made by using a multichannel pipette to transfer 10 µl of worm culture to 10 µl of single-worm lysis buffer containing proteinase K in a 96-well plate. The lysates were then processed, and genotyping PCRs and BamHI digests performed as described in the "Genotyping PCRs and restriction digestion" section in the main-text Methods. For wells with a hit, in order to identify homozygotes, 24 F2 progeny were transferred to single wells of a 96-well plate and incubated and screened as above.
For pooled screening of nhr-23::2xFLAG knock-ins, WT animals or lig-4(ok716) mutants were injected with 50 ng/µl of pJW1254 (nhr-23 PAM #1 targeting CRISPR/Cas9), 5 ng/µl of a myo-2::tdTomato co-injection marker, and 50 ng/µl of a sense nhr-23::2xFLAG 199mer (oligo #1719, Table S2). P0 animals were plated and marker positive wells were identified as above. All progeny from marker positive wells were washed out with 1 ml of M9+gelatin and diluted to ~10 worms/30µl and this volume (30 µl) was plated in 96 wells using a repeat pipetter. The number of worms/well was averaged in two rows of the plate to confirm the estimated concentration of 10 worms/well. Food was added, the plates were incubated, and lysates made as described above. Knock-in specific PCRs were performed using an oligo that internally bound the 2xFLAG sequence (#1715) and an oligo that bound external to the knock-in sequence.
Four rows were pooled for each PCR reaction with 0.5 µl of lysate from each row used in a 20 µl PCR. For wells with a hit, in order to identify homozygotes, 48-96 F2 progeny were transferred to single wells of a 96-well plate and incubated and screened as above.

Generation of lysates for immunoblotting
For the immunoblot in Figure 2, animals of the indicated genotype were synchronized by alkaline bleaching followed by plating overnight in the absence of food. Approximately 3000 arrested L1s were plated on 10 cm NGM-lite plates seeded with OP50 and incubated at 25ºC for 48 hours, at which point the animals were gravid adults. Animals were washed off of the plates with M9+gelatin, pelleted and transferred to a 1.5 ml tube and washed four times with 1 ml of M9. The M9 was aspirated, leaving 150 µl and the pellet flash-frozen in liquid nitrogen and stored at -80ºC. The pellet was resuspended by adding 150 µl 2xRIPA buffer (100 mM Tris-HCl, 900 mM NaCl, 2% NP-40, 1% Sodium deoxycholate and 0.2% SDS, pH 7.4) supplemented with Protease Inhibitor Cocktail Set III, EDTA-free (Calbiochem, #539134-1SET), 1mM PMSF, 10 µM MG-132 proteasome inhibitor (Caymon, #10012628), and 1 mM DTT. Worms were lysed by three cycles of sonication on ice (10 sec, 20% amplitude). Debris was pelleted by centrifugation at 14,000 rpm for 10 minutes at 4ºC and protein concentration was determined by a 660nm Protein Assay (Pierce). Four micrograms of total protein was resolved by SDS-PAGE using a Mini-PROTEAN TGX Stain-Free 4-15% gradient gel (Bio-Rad, #456-8086).
For the immunoblot in Figure 5, ten gravid adults were placed on 10 cm plates and incubated for four days at 25ºC.
Lysates were made by washing crowded, mixed stage animals off of these 10 cm plates in M9+gelatin, pelleting at 700xg for two minutes, and transferring to a 1.5 ml tube. The worms were washed four additional times with 1 ml of M9+gelatin. The M9+gelatin was aspirated to just above the worm pellet and the pellet was rapidly freeze-thawed three times (cycling between liquid nitrogen and a 42ºC water bath) before 4x Laemmli buffer was added to a final concentration of 1x. The samples boiled for 10 minutes, then the lysate was frozen for 15 minutes on dry ice and then boiled again for 10 minutes. Debris was pelleted by centrifugation at 14,000 rpm in a microcentrifuge for 5 minutes.

PEG/DMSO DH5a competent cells
A single DH5 alpha colony from a freshly struck plate was used to inoculate a 5 ml LB culture and and incubated overnight at 37ºC shaking at 225 rpm. This culture was used to inoculate 500 ml of LB which was shaken at 37ºC until an OD600 of 0.5-0.6 was reached. Cells were pelleted by centrifugation for 5 min at 2000 rpm, 4ºC. Cells were gently resuspended in 25 ml of ice cold TSB buffer (LB pH 6.1, 10% PEG-3350, 5% DMSO, 10 mM MgCl 2 , 10 mM MGSO 4 ), incubated on ice for 10 minutes, aliquoted, flash frozen in liquid nitrogen, and stored at -80ºC. To transform the cells, an aliquot was thawed on ice and the DNA to be transformed was mixed with 5xKCM (500 mM KCl, 150 mM CaCl 2 , 250 mM MgCl 2 ) and dH20 to a final volume of 100 µl at 1xKCM final concentration. An equal amount of cells was added, mixed by gentle inversion, and incubated on ice for 20 minutes. The mixture was then incubated at room temperature for 10 minutes before 1 ml of SOC or LB was added and the transformation was shaken for 1 hr at 37ºC before plating on LB containing appropriate antibiotics.     Figure  1D 80mer sense nhr-23::2xFLAG sense 16 3 0 0 pha-1(ts) OP50 Figure  1D 80mer antisense nhr-23::2xFLAG sense 47 3 0 0 pha-1(ts) OP50 Figure  1D 60mer sense nhr-23::2xFLAG sense 11 3 0 0 pha-1(ts) OP50 Figure  1D 200mer sense nhr-23::2xFLAG sense 28 10 3 2 pha-1(ts) OP50 Figure  1D 200mer sense nhr-23::2xFLAG antisense 57 4 0 0 pha-1(ts) HB101 Figure  3B 200mer sense n/a n/a n/a n/a n/a n/a 16 6 n/a PAM #2 29 bp 12 c 0 2 n/a 0 n/a n/a 0 1 n/a PAM #3 54 bp 8 d 0 0 0 0 1 0 0 0 1 e For the data presented in Figure 4, the number of animals with conversion events at the indicated PAMs, and number FLAG tag knock-in was presented. Here, a more detailed breakdown of the knock-in events is provided. There was no PAM #3 mutation present in the repair oligo used for the PAM #1 and PAM #2 sgRNA experiments.
a Pooled from all 200mer nhr-23-2xFLAG injections (Figures 1 and 3, Table 2). As these animals were selected for based on a potential FLAG insertion, there were no "PAM only" gene conversion events that would be identified in this dataset. b Includes 14 precise insertions and 8 partial insertions c Of the 12 sequenced animals, nine had no knocked-in sequence d Of the eight sequenced animals, six had no knocked-in sequence e 1 bp deletion in inserted 2xFLAG epitope


extfieldsofstudy: Biology Medicine

text:
Tat-Fused Recombinant Human SAG Prevents Dopaminergic Neurodegeneration in a MPTP-Induced Parkinson’s Disease Model

Excessive reactive oxygen species (ROS) generated from abnormal cellular process lead to various human diseases such as inflammation, ischemia, and Parkinson’s disease (PD). Sensitive to apoptosis gene (SAG), a RING-FINGER protein, has anti-apoptotic activity and anti-oxidant activity. In this study, we investigate whether Tat-SAG, fused with a Tat domain, could protect SH-SY5Y neuroblastoma cells against 1-methyl-4-phenylpyridinium (MPP+) and dopaminergic (DA) neurons in the substantia nigra (SN) against 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) toxicity. Western blot and immunohistochemical analysis showed that, unlike SAG, Tat-SAG transduced efficiently into SH-SY5Y cells and into the brain, respectively. Tat-SAG remarkably suppressed ROS generation, DNA damage, and the progression of apoptosis, caused by MPP+ in SH-SY5Y cells. Also, immunohistochemical data using a tyrosine hydroxylase antibody and cresyl violet staining demonstrated that Tat-SAG obviously protected DA neurons in the SN against MPTP toxicity in a PD mouse model. Tat-SAG-treated mice showed significant enhanced motor activities, compared to SAG- or Tat-treated mice. Therefore, our results suggest that Tat-SAG has potential as a therapeutic agent against ROS-related diseases such as PD.

INTRODUCTION
Reactive oxygen species (ROS) are generated during many cellular processes including catalysis and energy production (Bae et al., 2011;Zhu et al., 2004). In addition, abruptly overproduced ROS cause DNA damage, lipid peroxidation, cell death, and many pathological disorders such as inflammation, ischemia, diabetes, and Parkinson's disease (PD) (Ramalingam and . However, defense systems in cells remove excessively produced ROS and maintain the cellular balance of ROS (Ramalingam and Kim, 2012). PD, a neurodegenerative disease, is characterized by a progressive loss of dopaminergic (DA) neurons in the substantia nigra (SN) and an accumulation of Lewy bodies (Chinta and Andersen, 2008;Dawson and Dawson, 2003). DA neurons are associated with motor functions and loss of these neurons in PD patients could cause typical symptoms such as tremor, bradykinesia, rigidity, and akinesia (Miller et al., 2009).
Sensitive to apoptosis gene (SAG), a member of the zinc RING finger protein family, is found in both the cytoplasm and nucleus of the cell (Chanalaris et al., 2003). SAG can bind to metal ions, zinc or copper, and thereby protect cells from metal ion-induced apoptosis Swaroop et al., 1999). SAG overexpressed in U937 cells prevents ionizing radiationinduced apoptosis through inhibiting ROS production (Kim et al., 2011). It has been suggested that SAG can attenuate ischemia-induced brain injury in vivo mouse models (Yang et al., 2001) and in gerbil ischemia/reperfusion models (Chanalaris et al., 2003;Kim et al., 2010;Yoo et al., 2012). In addition, SAG overexpression promotes cell growth under serum starvation by mediating p27 ubiquitination and degradation . Elevated levels of SAG have been found in human colon carcinomas and transfection of antisense SAG into the tumor cells reduced growth of these cells .
Although many peptides and proteins have remarkable therapeutic potential, their low permeability and poor bioavailability have been obstacles in the process of developing them as drugs. Several useful strategies have been suggested to improve the delivery of proteins into various cells or tissues (Schwarze et al., 1999;van den Berg and Dowdy, 2011). Particularly, protein transduction domains (PTD) including Tat and PEP-1 have the ability to efficiently cross the blood brain barrier (BBB) and cellular membranes. Therefore, exogenous proteins can be efficiently delivered to viable tissues and cells through fusion with PTD (Ahn et al., 2010;Lee et al., 2012;Schwarze et al., 1999;van den Berg and Dowdy, 2011).
In this study, we investigated whether Tat-SAG fusion protein could be effectively delivered to SH-SY5Y neuroblastoma cells and brain tissues and so prevent 1-methyl-4-phenylpyridinium (MPP + )-induced apoptosis in SH-SY5Y cells, and protect DA neurons in the SN in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model. Therefore, we suggest that Tat-SAG may be a useful agent for treatment of ROS-related diseases including PD.

Materials and cell culture
Restriction endonuclease and T4 DNA ligase were purchased from Promega (USA). Plasmid pET-15b and Escherichia coli strain BL21 (DE3) were obtained from Novagen (Germany). Oligonucleotides were synthesized from Bioneer (Korea). An Ni 2+ -nitrilotriacetic acid Sepharose Superflow column was purchased from Qiagen (USA). Fetal bovine serum (FBS) and antibiotics were purchased from Gibco BRL (USA). MPTP was dissolved in saline and intraperitoneally (i.p.) injected. All other chemicals and reagents, unless otherwise stated, were obtained from Sigma-Aldrich (USA) and were of the highest analytical grade available.

Animals
Male C57BL/6 mice (8-week-old, Hallym University Experimental Animal Center, Korea) were used. They were housed at an ambient temperature of 23°C under a 12 h light /12 h dark cycle and were provided with food and water ad libitum. All experimental procedures involving animals and their care conformed to the Guide for the Care and Use of Laboratory Animals of the National Veterinary Research & Quarantine Service of Korea and were approved by the Hallym Medical Center Institutional Animal Care and Use Committee.

Preparation of Tat-SAG and SAG proteins and their transduction into SH-SY5Y cells
Expression vectors of Tat-SAG and SAG were constructed and two proteins were overexpressed and purified as described previously . To assess the dose-and timedependency of SAG and Tat-SAG transduction into SH-SY5Y cells, cells were plated into 6-well plates and were cultured in EMEM for 24 h. Cells were incubated with SAG and Tat-SAG in serum-free medium for 60 min. Then cells were harvested by trypsinization for 10 min and washed with phosphate-buffered saline (PBS) three times. The cells were lysed with a lysis buffer and Western blot analysis was performed. To evaluate the intracellular stability of Tat-SAG, cells were exposed to Tat-SAG (3 μΜ) for 60 min, and harvested at designated times (1-60 h). The levels of Tat-SAG were analyzed by Western blot analysis using an His antibody and a horseradish peroxidaseconjugated secondary antibody.

Cell viability measurement
An MTT assay was performed as described previously . SH-SY5Y cells were seeded into 96 well plates at the density of 10,000 cells/well overnight. The cells were treated with SAG or Tat-SAG in serum-free EMEM medium 1 h prior to incubation with MPP + (4.0 mM). After 18 h incubation, MTT was added to each well for 4 h and the formazan crystal was dissolved in DMSO. Optical density was measured at 590 nm using an ELISA plate reader.

ROS generation
ROS generation was assessed by determining fluorescence intensity derived from a process converting 2′,7′-dichlorofluorescein diacetate (DCF-DA) into fluorescent DCF. SH-SY5Y cells were plated into 96 well plates and pretreated with or without SAG and Tat-SAG for 60 min. This was followed by incubation with MPP + (4.0 mM) for 30 min and staining with DCF-DA (20 μM) for 30 min. The fluorescence was detected using a fluorescence microscope (Nikon, Japan) at 485 nm excitation and 538 nm emission.

Western blot analysis
Tissue samples were homogenized using a microtube homogenizer and cells were lysed in a lysis buffer. The protein concentrations in cell lysates or tissue homogenates were determined using a protein assay kit. Equal amounts of protein samples were resolved by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat dry milk in TBS-T buffer (25 mM Tris-HCl, 140 mM NaCl, 0.1% Tween 20, pH 7.5) and was incubated with a primary and secondary antibody. The membrane was developed with enhanced chemiluminescence reagents according to the manufacturer's instructions (USA). The β-actin level in each sample was used as a loading control. The band densities were analyzed by image analysis software.

Confocal fluorescence microscopy
After transduction of SAG and Tat-SAG into SH-SY5Y cells, their intracellular distributions were assessed by confocal fluorescence microscopy. Cells were grown on glass coverslips and exposed to SAG and Tat-SAG (3 μM) for 60 min. The cells were washed with PBS three times and fixed with 4% paraformaldehyde at room temperature for 10 min, followed by incubation with an anti-histidine antibody and Alexa fluor 488-conjugated secondary antibody (Invitrogen, USA). 4′6-diamidino-2phenylindole (DAPI) was used for nuclear counterstaining. The fluorescence image was analyzed using an Olympus FV-300 confocal fluorescence microscope.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay
To assess the effects of SAG or Tat-SAG on MPP + -induced DNA fragmentation, SH-SY5Y cells were pre-incubated with Tat, SAG, and Tat-SAG for 1 h, followed by treatment with MPP + (4.0 mM) for an additional 18 h. TUNEL staining was performed using a Cell Death Detection kit (Roche Applied Science, Switzerland) according to the manufacturer's instructions.

In vivo transduction of Tat fusion protein
To assess whether SAG and Tat-SAG proteins would traverse the BBB, SAG and Tat-SAG (3 mg/kg body weight) were i.p. injected into the mice (n = 7 mice/group). The mice were sacrificed 12 h later. The distribution of SAG and Tat-SAG in the SN were evaluated by histological analysis using a rabbit antihistidine polyclonal antibody and biotinylated goat anti-rabbit secondary antibody.

Protein treatment in MPTP-induced PD animal model
Mice were divided into 5 groups as follows; control, MPTP, MPTP + SAG, MPTP + Tat, and MPTP + Tat-SAG (n = 7 mice/ group). Saline, SAG, Tat, and Tat-SAG (3 mg/kg body weight) were i.p. administered to mice and then MPTP (20 mg/kg body weight) were injected four times at 2 h intervals the following day. The mice were sacrificed 7 days after MPTP treatment. Animals were anesthestized by an i.p. injection with sodium pentobarbital (100 mg/kg body weight) and fixed transcardially with 4% paraformaldehyde. The brains were post-fixed in the same fixative for 4 h, frozen and sectioned. Staining of tissue sections was carried out as previously described (Kim et al., 2008). Sections were blocked by 3% bovine serum albumin in PBS for 30 min and were probed with primary antibodies [His and tyrosine hydroxylase (TH)] and a biotinylated secondary antibody. Also, cresyl violet staining was performed to detect viable cells after TH immunostaining. The sections were visualized with 3,3′-diaminobenzidine (DAB) in 0.1 M Tris buffer and mounted on gelatin-coated slide. Images were captured and analyzed using an Olympus DP72 digital camera. The sections were processed and analyzed by an observer who was blinded with regard to mouse groups.

Behavior test
To assess the motor functions of mice, a Rotarod test was performed using Rotarod, Series 8 (IITC, USA) as described previously (Jeong et al., 2012). The acceleration of rotation gradually increased from 0 to 45 rpm. Each mouse was placed on the rotarod. The distance (m) and time (s) that each mouse spent on the rotarod without falling were automatically recorded.

Statistics
Data are expressed as the means ± SD. Comparison between groups was performed by Student's t-test. Values of P < 0.05, P < 0.01, P < 0.005, and P < 0.001 were considered to be statistically significant.

Tat-SAG transduced into SH-SY5Y cells in a time-and dose-dependent manner
Expression vectors of SAG and Tat-SAG, a SAG protein fused with a Tat peptide, were constructed and their schematic structures were shown in Fig. 1A. Two proteins were purified as described previously  and their purities were confirmed by SDS-PAGE and Western blot analysis (Figs. 1B and 1C). We first examined the abilities of SAG and Tat-SAG to penetrate into SH-SY5Y cells. SH-SY5Y cells were exposed to SAG and Tat-SAG (0.5-3.0 μM) for 60 min and the transduced protein levels in the cells were measured by Western blot analysis using an anti-His antibody. As shown in Fig. 2A, Tat-SAG levels increased in a time-dependent manner, compared to the level of SAG, which was not detected in the cells. In addition, Tat-SAG transduced into the cells in a time-dependent manner as observed when treated with Tat-SAG (3.0 μM) for various incubation times (10-60 min) (Fig. 2B). Immunofluorescence data revealed that Tat-SAG was significantly detected in the nucleus as well as cytoplasm of SH-SY5Y cells (Fig. 2C). We next evaluated the stability of Tat-SAG protein after its transduction into SH-SY5Y cells. Western blot analysis showed that the Tat-SAG level at 48 h was reduced to 24% of the initial level (1 h after transduction) (Fig. 2D). A significant level of Tat-SAG was observed in SH-SY5Y cells for 1-60 h after its transduction. These results suggest that Tat-SAG effectively transduced into SH-SY5Y cells and further, that the intracellular Tat-SAG level was significantly maintained for 48 h.
Transduced Tat-SAG inhibits ROS production and cell death SAG is known to scavenge ROS induced by redox agents . To confirm whether Tat-SAG would inhibit ROS generation, SH-SY5Y cells were treated with MPP + (4.0 mM) 30 min after exposure to SAG, Tat, or Tat-SAG for 60 min. SAG and Tat failed to suppress ROS generation caused by MPP + . In contrast, an obvious decrease in the fluorescent signal was observed in the MPP + + Tat-SAG treated sample (Fig.  3A). Next we assessed whether Tat-SAG could inhibit MPP +induced toxicity in SH-SY5Y cells using an MTT assay. The MTT assay showed that, when treated only with SAG or Tat-SAG, neither SAG nor Tat-SAG affected the proliferation of SH-SY5Y cells. However, when treated with MPP + , Tat-SAG (0.1-0.5 μM) significantly suppressed MPP + -induced toxicity in a dose-dependent manner (Fig. 3B). These results demonstrate that Tat-SAG significantly inhibited ROS production in response to MPP + and this was associated with suppression of MPP +induced toxicity. . To examine whether Tat-SAG suppresses DNA fragmentation caused by exposure to MPP + , a TUNEL assay was carried out after SH-SY5Y cells were treated with MPP + following transduction of SAG and Tat-SAG for 1 h. Transduction of Tat-SAG remarkably decreased the number of TUNEL labeled cells in contrast with the SAG and Tat treated samples (Fig. 4A). The percentage of fluorescently labeled cells in the Tat-SAG-treated sample was reduced to approximately 55%, compared with the MPP + -treated sample (Fig. 4B). Further, we investigated whether Tat-SAG could affect MPP +triggered apoptosis progression in SH-SY5Y cells. Tat-SAG markedly suppressed elevated levels of cleaved caspase-3 and bax, apoptotic markers, and also significantly up-regulated reduced levels of bcl-2, an anti-apoptotic marker (Figs. 4C and 4D). These findings demonstrate that transduced Tat-SAG inhibits MPP + -induced DNA fragmentation and apoptosis in SH-SY5Y cells, possibly by protecting SH-SY5Y neuroblastoma cells from oxidative stress caused by MPP + . for 18 h. Cell viability was assessed using an MTT assay. Effects of SAG and Tat-SAG on SH-SY5Y cell survival were compared in the presence or absence of MPP + . The bar graph represents means ± SD from three independent experiments. Data were analyzed by Student's t-test. *P < 0.05 and **P < 0.01 indicate a statistically significant difference between MPP + -treated control and Tat-SAG-treated samples.

A B C D Transduced Tat-SAG protects dopaminergic neurons in the SN from MPTP, a neurotoxin
We next evaluated the ability of Tat-SAG to efficiently cross the BBB as well as to enter SH-SY5Y cells. SAG and Tat-SAG were i.p. injected into mice (3 mg/kg body weight, n = 7 mice/ group) and mice brains were harvested 12 h later. Brain tissues were sectioned and stained using anti-histidine and biotinylated secondary antibodies. As shown in Fig. 5A, histological analysis showed that transduced Tat-SAG proteins were found in the, predominantly, cytoplasm as well as nucleus of DA neurons in the brain parenchyma, indicating that Tat-SAG has the capability to cross the BBB, in contrast with SAG. It has been shown that MPTP is converted to MPP + in glial cells and subsequently MPP + induces the death of DA neurons in the SN. We examined whether Tat-SAG can protect DA neurons in the SN against MPTP toxicity in a MPTP-induced PD mouse model. Mice (n = 7 mice/group) were i.p. injected with SAG and Tat-SAG (3 mg/kg body weight) and the next day MPTP (20 mg/kg body weight) was i.p. injected 4 times. The mice in the Tat-SAG group had significantly more TH positive cells in the SN, although less than the control mice, when compared to the MPTP-treated sample (Left panels, Fig. 5B). Furthermore, to confirm the survival of DA neurons in the SN of the mice after MPTP treatment, brain tissues were double-stained using a TH antibody and cresyl violet. Cresyl violet staining showed that MPTP treatment significantly induced the death of DA neurons in the SN. As expected, a significant number of viable TH neurons were seen in the Tat-SAG-treated sample (Right panels, Fig. 5B). The quantification data of TH positive cells shows that Tat-SAG protects TH positive neurons from MPTP (Fig. 5C). Next, to assess the effect of Tat-SAG on the motor functions in the MPTP-treated mice, we measured the distance and time that mice could stay on a moving rotarod without falling. The Tat-SAG-treated group spent significantly more time and moved longer distances on a rod, although less and shorter than the control group, compared to the MPTP, SAG, and Tat treated groups (Figs. 5D and 5E). Therefore, the results demonstrate that Tat-SAG represses the death of DA neurons in the SN caused by MPTP, thereby leading to a decrease in impairment of motor functions.

DISCUSSION
Neurodegenerative diseases, including Alzheimer's disease, Huntington's disease, and Parkinson's disease, are different from each other in terms of their causes and symptoms as well as the affected area of the brain (Chinta and Andersen, 2008;Dawson and Dawson, 2003;Miller et al., 2009;Ramalingam and Kim, 2012). However, excessive ROS generation seems to be tightly related to these neurodegenerative diseases. Mitochondria produce ROS and also neutralize ROS via the functions of various defense systems, which they possess, such as was quantified by TUNEL assay. *P < 0.05 and **P < 0.01 for MPP + versus other groups. (C, D) Effect of Tat-SAG on the levels of pro-or antiapoptotic markers. Levels of caspase-3, cleaved caspase-3, Bcl-2, and Bax were evaluated using (C) Western blot analysis and (D) were quantified using a densitometer. The following antibodies were used; mouse anti-caspase-3, mouse anti-cleaved caspase-3, mouse anti-bcl-2, mouse anti-bax, and mouse anti-β-actin. Western blotting analysis was independently repeated three times. Data were analyzed by Student's t-test. *P. < 0.05 and **P < 0.001 indicate a statistically significant difference between MPP + -treated group and other groups.
A C B D E glutathione, glutathione peroxidase, superoxide dismutase, and NADPH (Ramalingam and Kim, 2012). Therefore, it has been suggested that, if antioxidant molecules are up-regulated in target tissues or cells susceptible to oxidative damage, they may prevent oxidative stress and subsequent cell death. We produced PTD-fused proteins, including PEP-1-rpS3 and Tat-ANX, using PTD tools in order to increase therapeutic proteins and peptides in any cell type and tissues. These proteins, including PEP-1-rpS3 and Tat-ANX, can be efficiently delivered to cells and animal tissues, and they can inhibit ROS-related diseases involving inflammation and ischemic insult in animal models (Ahn et al., 2010;Lee et al., 2012). SAG, also known as RBX2/ROC2, a protein that is a component of the Skp1-Cullin1-F-box-protein E3 ubiqutin ligase (Tan et al., 2006b;Wei and Sun, 2010), was identified as a redoxinducible antioxidant protein Swaroop et al., 1999) and is induced by various stimuli, including redox agents, ischemia/reoxygenation, hypoxia, neurotoxin, heat shock, and UV irradiation (Wei and Sun, 2010). Human SAG protein possesses the binding sites for a lot of transcription factors, such as NF-κB, c-MYC, AP-1 and p53, and also it can bind to zinc and copper metal ions, which can induce apoptosis Jia et al., 2010). Numerous studies have shown that SAG functions as an antioxidant or anti-apoptotic protein. For example, SAG knockdown in human prostate cancer PC3 using siRNA, increased the susceptibility to inducers of ROS pro-duction or apoptosis, such as actinomycin D and etoposide . It is known that in multiple human tumor tissues, overexpression of SAG lead to reduced levels and half life of NOXA, a pro-apoptotic member of the Bcl-2 family. Also, SAG knockdown in cancer cells could sensitize the cells to radiation and induce apoptosis through accumulation of NOXA (Jia et al., 2010). SAG-overexpressing SH-SY5Y cells inhibited MPP + -induced neurotoxicity by down-regulation of ROS generation and c-Jun N-terminal kinase 1 activity (Kim et al., 2007). SAG overexpression in mouse epidermal cells promotes ultraviolet B-induced skin hyperplasia, but not skin tumors (He et al., 2008). In addition, SAG suppresses TPA-induced neoplastic transformation via ubiquitination and degradation of c-Jun (Gu et al., 2007) and protects cells from apoptosis by promoting the ubiquitination and degradation of procaspase-3 (Tan et al., 2006a). Thus, these reports suggested that an increased level of SAG could enhance survival of various cells.
As is well-known, in the intrinsic apoptotic pathway various apoptosis inducers, including ROS, induce the loss of mitochondrial membrane potential and release of pro-apoptotic proteins, such as Bax, into the cytosol. Consequently, it leads to activation of caspases and DNA fragmentation, a hallmarker of apoptosis. Consistent with other previous studies, we demonstrated that Tat-SAG significantly suppresses signaling of apoptosis including ROS generation (Fig. 3A), release of Bax, and activation of caspase-3 (Figs. 4C and 4D), thereby leading to sup- pression of DNA fragmentation (Figs. 4A and 4B) and cell death (Fig. 3B). Chanalaris et al. reported that SAG is found in both the cytoplasm and nucleus of rat cardiomyocytes (Chanalaris et al., 2003). Their findings were consistent with our results which showed that Tat-SAG was localized in the cytoplasm and nucleus of SH-SY5Y cells, although different from the cell line used in their studies. However, until now it was not evident that either Tat-SAG in the cytoplasm or nucleus can predominantly prevent MPP + -induced apoptosis or otherwise what role the transduced Tat-SAG can play in the nucleus of SH-SY5Y cells. Therefore, additional studies were required to clearly reveal the function of Tat-SAG in the SH-SY5Y cells.
Furthermore, our in vivo study revealed that Tat-SAG has the capability to cross the BBB and, through just a single administration of Tat-SAG, protects DA neurons in the SN against MPTP toxicity, subsequently maintaining the motor functions in the Tat-SAG-treated mice (Fig. 5). In this regard, we clearly showed that the increase of cellular levels of SAG enhances the cellular viability of the SH-SY5Y neuroblastoma cell line and DA neurons in the SN. Also, we previously reported that Tat-SAG significantly protects astrocyte cells and the CA1 region in the hippocampus of gerbil brains from ischemic damage .
The BBB is known as an obstacle to the delivery of therapeutic molecules to brain. It is therefore noteworthy that Tat-SAG, which has the capability to reduce oxidative stress-induced toxicity, could be effectively and easily delivered to cells and brain tissues, as shown in several studies including our data. On the other hand, we identified that PTD fusion proteins, including PEP-1-rpS3 and Tat-frataxin, are retained in cells and tissues for a considerable period and then gradually disappear (unpublished data). These results seem to demonstrate another advantage of PTD fusion proteins. Continuous overexpression in some cells and tissues could render excessive growthpromoting conditions to normal cells and tissues, thereby likely causing side effects. Consequently, our present study is considered as another promising observation showing that PTDfusion proteins could provide appropriate therapeutic agents against diseases such as PD.
In conclusion, the present study demonstrates that Tat-SAG can cross cellular membranes and the BBB, and suppress ROS generation, DNA damage, and cell death induced by oxidative stress and consequently protect DA neurons in the SN in a PD animal model. Taken collectively, it is likely that Tat-SAG could be a promising agent against ROS-related diseases such as inflammation and PD.