Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates


GCCATTTCAATAACGATATTTTCTGGCTTCTACCGCCCC ATTACTTTGACCAATTC L833TAG-F AGAATTAGATATTAATCGTTAGAGTGATTATGATGTCG ATC L833TAG-R GATCGACATCATAATCACTCTAACGATTAATATCTAAT TCT K929TAG-F GTTGAAACTCGCCAAATCACTTAGCATGTGGCACAAAT TTTG K929TAG-R CAAAATTTGTGCCACATGCTAAGTGATTTGGCGAGTTT CAAC L1004TAG-F CTGCTTTGATTAAGAAATATCCAAAATAGGAATCGGA GTTTGTCTATGGTG L1004TAG-R CACCATAGACAAACTCCGATTCCTATTTTGGATATTTC TTAATCAAAGCAG K1151TAG-F CTAGTGGTTGCTAAGGTGGAATAGGGGAAATCGAAGA AGTTAAA K1151TAG-R TTTAACTTCTTCGATTTCCCCTATTCCACCTTAGCAACC ACTAG K1153TAG-FW GTTGCTAAGGTGGAAAAAGGGTAGTCGAAGAAGTTAA AATCCGTT K1153TAG-R AACGGATTTTAACTTCTTCGACTACCCTTTTTCCACCTT AGCAAC D1361TAG-F GTCTTTATGAAACACGCATTTAGTTGAGTCAGCTAGGA GGTGA D1361TAG-R TCACCTCCTAGCTGACTCAACTAAATGCGTGTTTCATA AAGAC G1367TAG-FW CGCATTGATTTGAGTCAGCTAGGATAGGACCCCAAGA AGAAGAGGAAGGTG G1367TAG-

Fig. S1 .

 S1 Fig. S1. Peptide mass spec of G1367-AeF. 


Fig. S3 .

 S3 Fig. S3. Gene disruption activity of Cas9-ssODN conjugate in vitro and in cell. (a) Different Cas9 mutants were assembled into RNPs and incubated with DBCO or NH 2 modified ssODN. The resulting complex were measured for cleavage activity in vitro, and the cleavage products were measured on a 1% agarose. (b) Cas9 G1367-AeF mutant and wt Cas9 were assembled into RNP with DBCO or NH2 modified ssODN, the complex was delivered in HEK293 EGFP Teton cell through electroporation and analyzed by Flow cytometry 48h post-transduction. 


Fig. S4 .

 S4 Fig. S4. Cleavage activity of Cas9 mutants reacted with modified oligos in vitro and in cell. 




  (A) RNPs were pre-incubated with NH2 or DBCO modified oligo DNA, forming RNP-oligo DNA complex and analyzed for cleavage activity on agarose gel. (B) RNP with adaptor conjugated at different sites were transfected into HEK293-Teton-EGFP by RNAiMAX and analyzed by flow cytometry 2 days post-transduction. Error bars represent the standard deviation of triplicates. 


Fig. S5 .

 S5 Fig. S5. Oligonucleotide-adaptor paired with ssODN. Oligonucleotide-adaptor was mixed with ssODN at different molar ratio in vitro, and analyzed on a 2% agarose gel. 


Fig. S6 .

 S6 Fig. S6. Deep sequencing confirmation of HiBiT insertion and HDR enhancement. (A)Absolute HDR efficiency was quantified by deep sequencing. (B) HDR/NHEJ ratio was calculated based on data from deep sequencing. 


Fig

  Fig. S7. The HDR efficiency of RNP-dual ssODN complexes. RNP with adaptor conjugated at different sites were transfected into HEK293 by RNAiMAX and analyzed by Hibit assay 2 days post-transduction. Error bars represent the standard deviation of triplicates. Significance was calculated by means of a 2-tailed Student's t-test; * P < 0.05. 


Fig. S8 .

 S8 Fig. S8. Genotyping PCR of zygotes after RNP microinjection. A 141 bp fragment containing a 42 bp V5 tag can be specifically detected in edited zygotes using primer F2 and R2, and 99 bp band was amplified both in edited and unedited zygotes. 


Table S1 . Expression yield of Cas9 and its chemical mutants.

 S1 
	Mutation site	Expression yield	Mutation site	Expression yield
	WT	3mg/L	K929	0.2mg/L
	K3	Degradation	L1004	2.7mg/L
		seriously		
	D39	Inclusion body	K1151	2.8mg/L
	H41	0.25mg/L	K1153	1.5mg/L
	H754	0.17mg/L	G1361	0.8mg/L
	L833	Low expression	G1367	3mg/L
	Table S2. Primer and donor sequence used in the study	
	a. Primers used for mutagenesis PCR		
	Name	Sequence		
	K3TAG-FW	CCAAGAAGAAGAGGAAGGTGATGGATtAGAAATACTC
		AATAGGCTTAGATATCGGCAC	
	K3TAG-RV	GTGCCGATATCTAAGCCTATTGAGTATTTCTaATCCATC
		ACCTTCCTCTTCTTCTTGG	
	D39TAG-FW	GTTCAAGGTTCTGGGAAATACATAGCGCCACAGTATCA
		AAAAAAATC		
	D39TAG-RV	GATTTTTTTTGATACTGTGGCGCTATGTATTTCCCAGAA
		CCTTGAAC		
	H41TAG-FW	GTTCTGGGAAATACAGACCGCTAGAGTATCAAAAAAA
		ATCTTATAGGGGCTC		
	H41TAG-RV	GAGCCCCTATAAGATTTTTTTTGATACTCTAGCGGTCT
		GTATTTCCCAGAAC		
	H754TAG-FW	GAATTGGTCAAAGTAATGGGGCGGTAGAAGCCAGAAA
		ATATCGTTATTGAAATGGC	
	H754TAG-RV
Xinyu Ling, Bingteng Xie, Xiaoqin Gao, Liying Chang, Wei Zheng, Heqi Chen, Yujia Huang, Linzhi Tan, Mo Li, Tao Liu
Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates