Sniper2L is a high-fidelity Cas9 variant with high activity.pdf
Nature Chemical Biology | Volume 19 | August 2023 | 972–980 972 nature chemical biology Article https://doi.org/10.1038/s41589-023-01279-5 Sniper2L is a high-fidelity Cas9 variant with  high activity Young-hoon Kim1,2,3,4,20, Nahye Kim2,5,20, Ikenna Okafor6,20, Sungchul Choi2,  Seonwoo Min7, Joonsun Lee1, Seung-Min Bae1, Keunwoo Choi1, Janice Choi8,  Vinayak Harihar    8, Youngho Kim1, Jin-Soo Kim    9,  Benjamin P. Kleinstiver    10,11,12, Jungjoon K. Lee    1  , Taekjip Ha    8,13,14,15   &  Hyongbum Henry Kim    2,4,5,16,17,18,19  Although several high-fidelity SpCas9 variants have been reported, it has  been observed that this increased specificity is associated with reduced  on-target activity, limiting the applications of the high-fidelity variants  when efficient genome editing is required. Here, we developed an improved  version of Sniper–Cas9, Sniper2L, which represents an exception to this  trade-off trend as it showed higher specificity with retained high activity.  We evaluated Sniper2L activities at a large number of target sequences and  developed DeepSniper, a deep learning model that can predict the activity  of Sniper2L. We also confirmed that Sniper2L can induce highly efficient and  specific editing at a large number of target sequences when it is delivered  as a­ r­ib­onucleoprotein complex. Mechanically, the high specificity of  Sniper2L originates from its superior ability to avoid unwinding a target DNA  containing even a single mismatch. We envision that Sniper2L will be useful  when efficient and specific genome editing is required. Applications of SpCas9-induced genome editing are often restricted  due to off-target effects or insufficient on-target editing. Several  high-fidelity variants, such as eSpCas9(1.1)1, Cas9–HF12, HypaCas93,  Cas9_R63A/Q768A4, evoCas95, HiFi Cas96 and Sniper–Cas9 (referred  to in this manuscript as Sniper1)7, have been developed. However, the  modifications introduced in these variants to decrease off-target cleav- age also hamper their general on-target cleavage activities, such that  a trade-off between the general activity and specificity8 is observed  when the variants are tested with a large number of target sequences.  A high-fidelity variant that exhibits a general activity level similar to  that of SpCas9 would facilitate applications of SpCas9-based genome  editing in areas including gene therapy and genetic screening. In this study, we developed Sniper2L, a next-generation  high-fidelity variant, using directed evolution of Sniper1. To evaluate  Received: 11 May 2022 Accepted: 2 February 2023 Published online: 9 March 2023  Check for updates 1Toolgen, Seoul, Republic of Korea. 2Department of Pharmacology, Yonsei University College of Medicine, Seoul, Republic of Korea. 3Graduate Program  of Biomedical Engineering, Yonsei University College of Medicine, Seoul, Republic of Korea. 4Graduate Program of NanoScience and Technology, Yonsei  University, Seoul, Republic of Korea. 5Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine, Seoul, Republic  of Korea. 6Department of Biology,  Johns Hopkins University, Baltimore, MD, USA. 7LG AI Research, Seoul, Republic of Korea. 8Department of Biophysics,  Johns Hopkins University, Baltimore, MD, USA. 9Department of Biochemistry and NUS Synthetic Biology for Clinical & Technological Innovation (SynCTI),  National University of Singapore, Singapore, Singapore. 10Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA. 11Department  of Pathology, Massachusetts General Hospital, Boston, MA, USA. 12Department of Pathology, Harvard Medical School, Boston, MA, USA. 13Department of  Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, MD, USA. 14Department of Biomedical Engineering, Johns Hopkins University,  Baltimore, MD, USA. 15Howard Hughes Medical Institute, Baltimore, MD, USA. 16Center for Nanomedicine, Institute for Basic Science, Seoul, Republic  of Korea. 17Yonsei–Institute for Basic Science Institute, Yonsei University, Seoul, Republic of Korea. 18Severance Biomedical Science Institute, Yonsei  University College of Medicine, Seoul, Republic of Korea. 19Institute for Immunology and Immunological Diseases, Yonsei University College of Medicine,  Seoul, Republic of Korea. 20These authors contributed equally: Young-hoon Kim, Nahye Kim, Ikenna Okafor.   e-mail: jj.lee@toolgen.com; tjha@jhu.edu;  hkim1@yuhs.ac Nature Chemical Biology | Volume 19 | August 2023 | 972–980 973 Article https://doi.org/10.1038/s41589-023-01279-5 of on-target indels with many different single-guide RNAs (sgRNAs)  compared with Clone-2 and Clone-3, which showed low on-target  indel efficiencies with the same sgRNAs. High indel frequencies were  observed when these variants were tested with the sgRNA EMX1.3,  which was used in the Sniper screen. To distinguish SpCas9 variants  with reduced on-target activities, such as Clone-2 and Clone-3, from  those with maintained on-target activities, we needed to perform the  Sniper screen with an sgRNA that would result in low on-target indel effi- ciencies with Clone-2 and Clone-3 while retaining wild-type (WT)-level  indel efficiencies with Clone-1. When we used EMX1.6 sgRNA, which  was previously used to determine the specificity of SpCas9 (ref. 9), we  found that the on-target activities of Clone-2 and Clone-3 were dra- matically decreased as compared with that of Clone-1 (Supplementary   Fig. 2). Thus, we chose EMX1.6 sgRNA for screening in the current study.  Because the mismatches in the previous Sniper screen were at positions  5–7 (proximal to the PAM) and positions 17 and 18 (distal to the PAM),  we attempted to make a mismatch in the previously untested middle  region, which spans positions 8–16. The center of the middle region  would include positions 11–13 or 10–14. Among these positions, a previ- ous study showed that the induction of C to U mutations at position 13  of an EMX1.6 sgRNA resulted in the highest relative cleavage efficiency9.  In addition, this mismatch induces wobble base pairing, which gener- ally results in high relative activities at mismatched targets (that is,  low specificity) by SpCas9 and its variants8. Thus, as the sgRNA and  the specificity and activity of Sniper2L at a large number of target  sequences, we delivered it together with guide RNA (gRNA) using two  different methods: lentiviral expression and electroporation of ribo- nucleoprotein (RNP) complexes, a therapeutically relevant method.  Our high-throughput evaluations showed that Sniper2L exhibits higher  fidelity than Sniper1 while retaining its general level of activity, similar  to that of SpCas9, overcoming the trade-off between activity and speci- ficity regardless of the delivery method. We believe that Sniper2L will  facilitate applications of genome editing due to its high general activity  and low levels of off-target effects. Results Directed evolution of Sniper1 Previously, we used ‘Sniper screen’ for directed evolution of SpCas9  in Escherichia coli (E. coli)7 (Supplementary Fig. 1). In brief, both posi- tive (SpCas9-mediated cleavage of a plasmid containing a lethal gene  (ccdB)) and negative (lack of E. coli-killing cleavage at a mismatched  off-target genomic site) selection pressure were applied to SpCas9  mutant libraries, in which the entire SpCas9-encoding sequence con- tained random errors (library complexity, up to 107); a fragment of the  human EMX1 gene was used for the matched and mismatched target  sequences. The initial Sniper screen resulted in the identification of  three SpCas9 variants named Clone-1, Clone-2 and Clone-3 (ref. 7). We  selected Clone-1 (that is, Sniper1) because it induced high frequencies  a b c P = 0.032 P = 0.014 0 1 2 3 4 Relative indel frequency (normalized to SpCas9) Relative indel frequency (normalized to SpCas9) 2.0 1.5 1.0 0.5 0 SpCas9 Indel frequencies (%) Specificity On target Of target 0 0.4 0.5 0.6 0.7 0.8 20 40 60 Sniper1 SpCas9 Sniper1 E1007A E1007R E1007A E1007R E1007L E1007P E1007S E1007Y SpCas9 Sniper1 E1007A E1007R E1007L E1007P E1007S E1007Y E1007N ZSCAN2 E1007D E1007C E1007Q E1007G E1007H E1007I E1007L E1007K E1007M E1007F E1007P E1007S E1007T E1007W E1007Y E1007V Fig. 1 | Schematics for hit identification using Sniper screen and hit  optimization using site saturation mutagenesis. a, Indel frequencies at on- target (blue) and off-target (orange) sequences and specificities determined  after transfection of plasmids encoding SpCas9 or Sniper1 variants into HEK293T  cells. Sniper1 variants were generated by site saturation mutagenesis at the  1,007th amino acid codon (originally a Glu codon); the resulting amino acids  at that position are shown on the x axis. Indel frequencies and specificities  are shown on the left and right y axes, respectively. Specificity was calculated  as 1 − (indel frequencies at off-target sequences divided by those at on-target  sequences). The averages of three replicates are indicated by dark blue and red  horizontal lines. The name of the gene in which the target sequence is located is  indicated at the top of the graph. The number of independent transfections (n)  is n = 3. Statistical significances are shown (no statistical significance (P > 0.05)  unless specified in the figure; Kruskal–Wallis test). b,c, Indel frequencies induced  by SpCas9 and Sniper1 variants based on plasmid delivery at on-target (b) and  off-target (c) sequences in HEK293T cells. The results for each target sequence  are shown in Supplementary Fig. 5. The boxes represent the 25th, 50th and  75th percentiles; whiskers show the 10th and 90th percentiles. The number of  analyzed target sequences n = 8.
Title: Sniper2L is a high-fidelity Cas9 variant with high activity ​
Authors: Young-hoon Kim, Nahye Kim, Ikenna Okafor, Sungchul Choi, Seonwoo Min, Joonsun Lee, Seung-Min Bae, Keunwoo Choi, Janice Choi, Vinayak Harihar, Youngho Kim, Jin-Soo Kim, Benjamin P. Kleinstiver, Jungjoon K. Lee, Taekjip Ha, Hyongbum Henry Kim ​

Publisher: Nature Chemical Biology
Date: 9 March 2023 ​
Abstract: Although several high-fidelity SpCas9 variants have been reported, it has been observed that this increased specificity is associated with reduced on-target activity, limiting the applications of the high-fidelity variants when efficient genome editing is required. ​ Here, we developed an improved version of Sniper–Cas9, Sniper2L, which represents an exception to this trade-off trend as it showed higher specificity with retained high activity. ​ We evaluated Sniper2L activities at a large number of target sequences and developed DeepSniper, a deep learning model that can predict the activity of Sniper2L. ​ We also confirmed that Sniper2L can induce highly efficient and specific editing at a large number of target sequences when it is delivered as a ribonucleoprotein complex. ​ Mechanically, the high specificity of Sniper2L originates from its superior ability to avoid unwinding a target DNA containing even a single mismatch. ​ We envision that Sniper2L will be useful when efficient and specific genome editing is required. ​