A Cas9–transcription factor fusion protein enhances homology-directed repair efficiency

Abstract

Precise gene insertion or replacement in cells and animals that requires incorporation of a foreign DNA template into the genome target site by homology-directed repair (HDR) remains an inefficient process. One of the limiting factors for the inefficiency of HDR lies in the limited chance for colocalization of the donor template and target in the huge genome space. We here present a strategy to enhance HDR efficiency in animal cells by spatial and temporal colocalization of the donor and Cas9 by coupling the CRISPR system with a transcription factor (TF). We first identified that THAP domain-containing 11 (THAP11) can coordinate with CRISPR/Cas9 to increase HDR stably through screening multiple TFs from different species. We next designed donor structures with different fusion patterns with TF-specific DNA-binding motifs and found that appending two copies of THAP11-specific DNA binding motifs to both ends of the double-stranded donor DNA has an optimal effect to promote HDR. The THAP11-fused CRISPR system achieved more than twofold increase in HDR-mediated knock-in efficiency for enhanced green fluorescent protein (EGFP) tagging of endogenous genes in 293T cells. We also demonstrated up to 6-fold increases of knock-in through the combinational use of the TF-fused CRISPR and valnemulin, a recently discovered small-molecule HDR enhancer. This modified CRISPR system provides a simple but highly efficient platform to facilitate CRISPR-mediated KI manipulations. Precise gene insertion or replacement in cells and animals that requires incorporation of a foreign DNA template into the genome target site by homology-directed repair (HDR) remains an inefficient process. One of the limiting factors for the inefficiency of HDR lies in the limited chance for colocalization of the donor template and target in the huge genome space. We here present a strategy to enhance HDR efficiency in animal cells by spatial and temporal colocalization of the donor and Cas9 by coupling the CRISPR system with a transcription factor (TF). We first identified that THAP domain-containing 11 (THAP11) can coordinate with CRISPR/Cas9 to increase HDR stably through screening multiple TFs from different species. We next designed donor structures with different fusion patterns with TF-specific DNA-binding motifs and found that appending two copies of THAP11-specific DNA binding motifs to both ends of the double-stranded donor DNA has an optimal effect to promote HDR. The THAP11-fused CRISPR system achieved more than twofold increase in HDR-mediated knock-in efficiency for enhanced green fluorescent protein (EGFP) tagging of endogenous genes in 293T cells. We also demonstrated up to 6-fold increases of knock-in through the combinational use of the TF-fused CRISPR and valnemulin, a recently discovered small-molecule HDR enhancer. This modified CRISPR system provides a simple but highly efficient platform to facilitate CRISPR-mediated KI manipulations. The CRISPR/Cas9-induced precise genetic modification through homology-directed repair (HDR) is a desired process in many applications, but HDR is not readily performed for its low efficiency. The strategy enhancing HDR efficiency could benefit many HDR-involved applications, such as the creation of gene knock-in (KI) animals and gene therapy for correction of genetic mutations. HDR is a DNA recombination process that repairs double-strand break using a homologous donor as the repair template, thus requires a spatial and temporal colocalization of the template and target (1Jasin M. Rothstein R. Repair of strand breaks by homologous recombination.Cold Spring Harb. Perspect. Biol. 2013; 5a012740Crossref PubMed Scopus (549) Google Scholar). It is anticipated that the donor searching for a homologous sequence is a key rate-limiting step for HDR occurrence because of the limited chance for coupling of the donor and target in the huge genome space. Therefore, increasing a local donor DNA concentration near the CRISPR/Cas9 cleavage site instead of allowing a randomly floating donor in the nucleoplasm could enhance the HDR frequency (2Ma M. Zhuang F. Hu X. Wang B. Wen X.Z. Ji J.F. Xi J.J. Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-avidin/biotin-donor DNA system.Cell Res. 2017; 27: 578-581Crossref PubMed Scopus (59) Google Scholar). This idea has been verified by studies using different strategies to physically position donor DNA near the cleavage site (2Ma M. Zhuang F. Hu X. Wang B. Wen X.Z. Ji J.F. Xi J.J. Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-avidin/biotin-donor DNA system.Cell Res. 2017; 27: 578-581Crossref PubMed Scopus (59) Google Scholar, 3Lee K. Mackley V.A. Rao A. Chong A.T. Dewitt M.A. Corn J.E. Murthy N. Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering.Elife. 2017; 6e25312Crossref PubMed Google Scholar, 4Savic N. Ringnalda F.C. Lindsay H. Berk C. Bargsten K. Li Y. Neri D. Robinson M.D. Ciaudo C. Hall J. Jinek M. Schwank G. Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair.Elife. 2018; 7e33761Crossref PubMed Scopus (89) Google Scholar, 5Aird E.J. Lovendahl K.N. St Martin A. Harris R.S. Gordon W.R. Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template.Commun. Biol. 2018; 1: 54Crossref PubMed Scopus (131) Google Scholar, 6Gu B. Posfai E. Rossant J. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos.Nat. Biotechnol. 2018; 36: 632-637Crossref PubMed Scopus (143) Google Scholar, 7Ling X. Xie B. Gao X. Chang L. Zheng W. Chen H. Huang Y. Tan L. Li M. Liu T. Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates.Sci. Adv. 2020; 6eaaz0051Crossref PubMed Scopus (47) Google Scholar, 8Carlson-Stevermer J. Abdeen A.A. Kohlenberg L. Goedland M. Molugu K. Lou M. Saha K. Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing.Nat. Commun. 2017; 8: 1711Crossref PubMed Scopus (94) Google Scholar). In general, chemical modification of the donor DNA is required to mediate an attachment or binding between the donor and CRISPR components, such as using biotinylated donor and streptavidin-fused Cas9 protein to colocalize them (2Ma M. Zhuang F. Hu X. Wang B. Wen X.Z. Ji J.F. Xi J.J. Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-avidin/biotin-donor DNA system.Cell Res. 2017; 27: 578-581Crossref PubMed Scopus (59) Google Scholar, 6Gu B. Posfai E. Rossant J. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos.Nat. Biotechnol. 2018; 36: 632-637Crossref PubMed Scopus (143) Google Scholar, 8Carlson-Stevermer J. Abdeen A.A. Kohlenberg L. Goedland M. Molugu K. Lou M. Saha K. Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing.Nat. Commun. 2017; 8: 1711Crossref PubMed Scopus (94) Google Scholar), increasing the probability of the use of the donor as a repair template. We here devised a simple system by using a nonchemically modified donor structure to colocalize donor and CRISPR components. The transcription factor (TF) controls gene transcription by binding to a specific DNA sequence, thereby can be exploited to recruit specific DNA in the presence of recognized DNA sequence (9Spitz F. Furlong E.E. Transcription factors: From enhancer binding to developmental control.Nat. Rev. Genet. 2012; 13: 613-626Crossref PubMed Scopus (1169) Google Scholar). For this purpose, we constructed a fusion protein between Cas9 and TF DNA-binding domain (DBD) and appended the TF-recognized DNA sequence to the ends of donor to co-localize the two components in DNA repair process, with the aim to increase the HDR efficiency after CRISPR cleavage. This design is theoretically feasible, but some factors could affect its performance. For examples, is there sufficient affinity between the TF and DNA motif to realize a local enrichment of the donor DNA in the CRISPR cleavage site; how many and where are the binding motifs fused to the donor DNA; do the redesigned/fused protein and DNA structure affect the affinity and Cas9 cleavage activity? Therefore, identifying the TFs and binding motifs applicable in the CRISPR-mediated HDR system and optimizing their structures to realize an effective cooperation between them are critical. In this study, we find that a TF, THAP domain-containing 11 (THAP11), can couple with the CRISPR system to effectively enhance HDR frequency (2–3 folds) when using a TF-binding motif-appended DNA donor. We also try a combinational use of the TF-fused CRISPR and small-molecule treatment and further increase the HDR frequency to 2- to 6-folds in dependence on the cell lines and genome sites tested. A total of 8 TFs, including human-, mouse-, and pig-derived activating transcription factor 3 (ATF3) and THAP11, as well as yeast GAL4 and PDR1, were tested with respect to their capacity to increase HDR in animal cells. We fused DBDs of these TFs (Table 1) to the C terminal of humanized spCas9. For the donor, we used a linear dsDNA with a 200-bp left homology arm and 800-bp right homology arm to insert a T2A–enhanced green fluorescent protein (EGFP) tag into the 3’ end of GAPDH gene, and two copies of TF-binding motifs were added to both ends of the dsDNA donor (Fig. 1A). The sequence information of TF-binding motifs was obtained from JASPAR 2020 (10Fornes O. Castro-Mondragon J.A. Khan A. van der Lee R. Zhang X. Richmond P.A. Modi B.P. Correard S. Gheorghe M. Baranašić D. Santana-Garcia W. Tan G. Chèneby J. Ballester B. Parcy F. et al.JASPAR 2020: Update of the open-access database of transcription factor binding profiles.Nucleic Acids Res. 2020; 48: D87-D92PubMed Google Scholar) (Table 2). A predicted human ATF3 or THAP11-recognized sequence was used for all human, mouse, and pig TF orthologs. After cotransfection of Cas9–DBD/gRNA and donors harboring corresponding binding motifs, the HDR efficiency was determined by the EGFP tagging rate that successful tagging enables a fused GAPDH–EGFP expression. In all tested TFs, we found that only human- and mouse-derived THAP11 showed significant HDR-promoting effect (Fig. 1B). We further detected if different appending terminals of binding motif affect the HDR efficiency. Two copies of motifs were appended to either or both ends of dsDNA donors, and results demonstrated that a donor containing THAP11-binding motifs at both ends (LR) significantly and stably increased HDR efficiency when cotransfection with Cas9 fused with any of human, mouse, and pig THAP11–DBDs. However, the donor containing the same motifs in the right arm (R) only increased HDR in the presence of Cas9 fused with the DBD from mouse THAP11 (mTHAP11), and no effect was observed from the donor containing the motifs in left arm (L). Besides, coupled use of mouse ATF3 DBD-fused Cas9 and donor with ATF3 binding motifs at both ends also increased HDR, but the increase was not as prominent as that observed in THAP11 (Fig. 1C). We also performed Western to the EGFP in and found the prominent EGFP when using the donor with two motifs, by donors with motifs at the right end and left end (Fig. in with the HDR efficiency detected by in modified of the donor increased EGFP with the donor binding motif (Fig. We next if increased of the binding motif in the donor can increase HDR efficiency. this purpose, we the donors with two and copies of THAP11 motifs at both ends in the showed that copies of motifs increased cells with two copies of motifs in the donor, but the increase was (Fig. We also performed and that the cells used to HDR rate were all from HDR-mediated KI of the donor into the genome (Fig. results that THAP11 is well applicable in the CRISPR/Cas9 system to enhance HDR, and the optimal effect can be achieved from coupled use of Cas9 fused with DBD from human THAP11 or and the donor with two copies of motifs at both of DNA-binding of TFs used in this of DNA-binding transcription in a site motifs of TFs used in this site TF binding motifs to JASPAR 2020 of TF used for of binding transcription TF binding motifs to JASPAR 2020 (10Fornes O. Castro-Mondragon J.A. Khan A. van der Lee R. Zhang X. Richmond P.A. Modi B.P. Correard S. Gheorghe M. Baranašić D. Santana-Garcia W. Tan G. Chèneby J. Ballester B. Parcy F. et al.JASPAR 2020: Update of the open-access database of transcription factor binding profiles.Nucleic Acids Res. 2020; 48: D87-D92PubMed Google The of TF used for of binding in a TFs, transcription TFs, transcription The a Cas9 structure with HDR by fusion of Cas9 and the donor two copies of THAP11-binding motifs at both ends to an effective The of Cas9 fusion used in the is Cas9 fused with an at the C terminal (Fig. can be used as the to the efficiency to HDR efficiency. We also tested system for EGFP tagging at the 3’ end of gene by HDR in 293T cells and found a increase in cells and increase in HDR efficiency by of the rate with using Cas9 and the donor (Fig. fusion with the cleavage of we used a to the targeted cleavage of the modified CRISPR The a EGFP with a CRISPR cleavage site by two (Fig. between the two enables an recombination to an EGFP sequence, the that can be by to the CRISPR cleavage We found that both Cas9 and effectively the target site to the cells with a rate (Fig. This that fusion not the targeted cleavage of the of the TF-fused CRISPR in HDR, we the of the key factors in DNA repair between Cas9 and 293T cells. results showed that the and and HDR and were not between Cas9 and that not promote HDR through the DNA repair (Fig. to TF the donor DNA from by their affinity to promote HDR, of the donor DNA in 293T cells showed no significant between the and fused CRISPR system (Fig. The total donor is thus not in the presence of the TF-fused We the with a harboring a in the C The CRISPR its in increasing HDR with the CRISPR (Fig. if TF fusion increases the binding between the CRISPR and donor, we used to the affinity between the TF-fused CRISPR and donor. The showed a significantly increased binding efficiency between the CRISPR and donor after fusion with the CRISPR no increasing effect to their binding (Fig. In we can an colocalization of donor DNA and protein in the of the cells through (Fig. that the TF-fused CRISPR HDR efficiency through colocalization of the donor and the CRISPR by the affinity between the TF and its binding treatment is a simple to increase HDR in animal cells J. D. J. T. Chen Zhang J. enhances and knock-in Commun. PubMed Scopus Google Scholar, T. B. W. S. K. R. Increasing the efficiency of homology-directed repair for precise gene editing in Biotechnol. PubMed Scopus Google Scholar, T. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by of end Biotechnol. PubMed Scopus Google Scholar, S. J.A. homology-directed human genome by of CRISPR/Cas9 PubMed Scopus Google Scholar). We here also to use to further increase HDR efficiency. small-molecule a or effect to promote HDR S. T. repair with increases precise genome editing in Commun. 2018; PubMed Scopus Google Scholar, Li Li Chen W. C. D. Zhang L. Wen W. J. N. W. T. Zhang Efficient precise with a HDR donor after double-stranded DNA Biol. 2017; PubMed Scopus Google Scholar). used small-molecule HDR are also thereby a effect in in cells or with the HDR-promoting effect but a and and found a of HDR two and or and valnemulin, HDR-promoting were further tested in the present as a is a and is an for use L. M. R. T. L. The of in the of by of with PubMed Scopus (59) Google Scholar). In a for the HDR-promoting showed its effect at the effect at the of to (Fig. use of and can increase HDR with either of them (Fig. the of the two at their found that not affect cell and but cell and increased cell in 293T with controls (Fig. C and The effect of on CRISPR cleavage was tested by the (Fig. After CRISPR and treatment in 293T no significant was found in the CRISPR cleavage by a cleavage efficiency (Fig. and of alleles (Fig. between treatment and the combinational effect of and on increasing HDR, 293T cells were with the CRISPR containing and and donors for EGFP tagging at and with for HDR efficiency through found that the use of increased HDR efficiency and at GAPDH and with Cas9 (Fig. A and treatment and increased efficiency for the two (Fig. A and an benefit for of HDR in the TF-fused CRISPR We also tested the of and in cell lines and found increase in HDR efficiency. in the EGFP tagging system at GAPDH and 2- and increased HDR efficiency was found in cells (Fig. and increase for both was achieved in cells (Fig. HDR-promoting were than using either of or Increasing local concentration of donor DNA at the CRISPR/Cas9 cleavage site is a to promote HDR efficiency. achieved significantly increased HDR in animal cells and via multiple (2Ma M. Zhuang F. Hu X. Wang B. Wen X.Z. Ji J.F. Xi J.J. Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-avidin/biotin-donor DNA system.Cell Res. 2017; 27: 578-581Crossref PubMed Scopus (59) Google Scholar, 3Lee K. Mackley V.A. Rao A. Chong A.T. Dewitt M.A. Corn J.E. Murthy N. Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering.Elife. 2017; 6e25312Crossref PubMed Google Scholar, 4Savic N. Ringnalda F.C. Lindsay H. Berk C. Bargsten K. Li Y. Neri D. Robinson M.D. Ciaudo C. Hall J. Jinek M. Schwank G. Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair.Elife. 2018; 7e33761Crossref PubMed Scopus (89) Google Scholar, 5Aird E.J. Lovendahl K.N. St Martin A. Harris R.S. Gordon W.R. Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template.Commun. Biol. 2018; 1: 54Crossref PubMed Scopus (131) Google Scholar, 6Gu B. Posfai E. Rossant J. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos.Nat. Biotechnol. 2018; 36: 632-637Crossref PubMed Scopus (143) Google Scholar, 7Ling X. Xie B. Gao X. Chang L. Zheng W. Chen H. Huang Y. Tan L. Li M. Liu T. Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates.Sci. Adv. 2020; 6eaaz0051Crossref PubMed Scopus (47) Google Scholar, 8Carlson-Stevermer J. Abdeen A.A. Kohlenberg L. Goedland M. Molugu K. Lou M. Saha K. Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing.Nat. Commun. 2017; 8: 1711Crossref PubMed Scopus (94) Google Scholar). For examples, using an Cas9 to recruit donor to by homology showed to KI efficiency in mouse no KI editing was observed in all tested genes for the CRISPR and donor system (2Ma M. Zhuang F. Hu X. Wang B. Wen X.Z. Ji J.F. Xi J.J. Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-avidin/biotin-donor DNA system.Cell Res. 2017; 27: 578-581Crossref PubMed Scopus (59) Google Scholar). strategy achieved to KI efficiency for dsDNA KI in two mouse with to KI efficiency when using the CRISPR system B. Posfai E. Rossant J. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos.Nat. Biotechnol. 2018; 36: 632-637Crossref PubMed Scopus (143) Google Scholar). et a modified Cas9 protein containing of donor DNA by of thereby the donor template to the cleavage of and a HDR increase as with in cells. using a DNA to donor by modified increased HDR efficiency as with or X. Xie B. Gao X. Chang L. Zheng W. Chen H. Huang Y. Tan L. Li M. Liu T. Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates.Sci. Adv. 2020; 6eaaz0051Crossref PubMed Scopus (47) Google Scholar). studies that the CRISPR cleavage and donor template can a increase in HDR efficiency. on the we the CRISPR and donor by the affinity between the TF and its binding We multiple of TFs and binding motifs and found that THAP11 of different in fusion with Cas9 protein all increased CRISPR-mediated HDR. The effect can be achieved in cotransfection of THAP11 DBD-fused Cas9 and dsDNA donor with two copies of the binding motif at both However, the donor with binding motif not significantly increase HDR that increased of binding motifs at the donor increase affinity between the donor and TF-fused the tested TFs, also to increase HDR, but TFs, and showed no effect on HDR an in TF between yeast and with that used modified Cas9 and donor modified and in the CRISPR system and The modified CRISPR can be used in and the modified donor can be by system on the dsDNA donor, HDR with a template, thereby the for KI manipulations. of this is that the TF-fused system can be with to further increase HDR efficiency. The identified were thus a in animal or In is an that protein in by binding in L. M. R. T. L. The of in the of by of with PubMed Scopus (59) Google Scholar). its in cells. We also observed that has no in 293T by cell and with results the of using in animal and for KI combinational use of TF-fused CRISPR/Cas9 system and valnemulin, 6-fold increase in the KI rate can be found in 293T with only the use of The combinational use also increased KI to and in and a HDR-promoting effect of system in cell In we a TF-fused CRISPR/Cas9 system as a platform that an enhanced KI in animal cells. This system is applicable in multiple cell and can in with small-molecule treatment to promote KI efficiency. The Cas9 gene was constructed by fusion of DBDs of TFs THAP11, and to the 3’ terminal of humanized with a between of efficiency on HDR, we also a that to by a The genome editing efficiency can be by to the are dsDNA containing an sequence by homology and two or copies of specific TF-binding motifs at the ends of homology of the donor and Cas9 into animal cells can mediate a precise insertion of to the end of specific gene or an fusion were by the system The 293T cells were in modified with at in containing cell were from For KI 293T cells were in and with to the For and both are of were added into the cell at after and cells were further for for The of and are and for cell a concentration and in KI or HDR efficiency of EGFP tagging at specific genes was detected by the cells by after or treatment as In of using including fusion cells were also to present efficiency. KI efficiency was by the EGFP rate to Western was performed as H. Wang G. H. R. Liu T. Wang Liu Y. B. D. Huang J. Y. Li S. X. et in Res. PubMed Scopus Google Scholar). In 293T cells were using and the was by The with an of the total protein was on for protein protein in was the The target were with of EGFP and by with The protein was using and using an Precise KI of the sequence into or GAPDH was verified by and For identifying the the homology arm but in the the homology arm were used (Fig. was performed to a X. Li H. Liu D. G. Li G. J. Wang D. C. Wang H. Y. J. Zheng E. F. Zhang M. et with enhanced and 2018; PubMed Scopus Google Scholar). DNA from KI 293T cells was with to the sites of to and KI for and GAPDH with different The was a through of the EGFP was performed to the of and HDR key RNA of cells and donor were using the and into as through transcription using the with was performed using and The of gene was by the to endogenous and as the as with the Cas9 DNA to the was with and into 293T cells with the CRISPR harboring Cas9 or and after cells were with with in and with to Cas9 The cells were with the and with The fluorescent was an to the of Cas9 and the donor. We used to the binding between protein and DNA donor. was performed the In 293T cells were with DNA donor and CRISPR containing and GAPDH and The cells were and to at after were by and protein was by and the DNA was and used as the template for The DNA was used as the The were used for of the donor and GAPDH donor, and were also used to or GAPDH donors of DNA for CRISPR cleavage efficiency was determined by the or K. A. Efficient correction in human cells using CRISPR-Cas9 PubMed Scopus Google Scholar). The is an HDR after of a double-strand break F. J.E. of recombination by double-strand breaks in Biol. Rev. PubMed Google Scholar). We a site by two sequence in the same into EGFP by a human enhancer and After CRISPR repair of the two an EGFP repair is a highly efficient DNA repair process, cleavage efficiency can be determined by cells with For the the was by after CRISPR-mediated gene The were and to can be by as repair of breaks a of different at the target was added into the DNA for of CRISPR cleavage efficiency was by the of can be by in the total in cell after treatment were by using the cells were in and with and at the for was with of the and of the was added to the to cell were for at and was at using a after small-molecule treatment was by of and can cells and After cells were with and were with are as the and from of are as or on in the in was determined by for two and with for or more were significant at are the This The that no of with the of this G. H. and W. the G. H. H. and X. designed and performed the H. Y. and G. L. and the the results and the of the This was in by the of

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