Engineered FnCas12a with enhanced activity through directional evolution in human cells

Abstract

Clustered regularly interspaced short palindromic repeat–Cas12a has been harnessed to manipulate the human genome; however, low cleavage efficiency and stringent protospacer adjacent motif hinder the use of Cas12a-based therapy and applications. Here, we have described a directional evolving and screening system in human cells to identify novel FnCas12a variants with high activity. By using this system, we identified IV-79 (enhanced activity FnCas12a, eaFnCas12a), which possessed higher DNA cleavage activity than WT FnCas12a. Furthermore, to widen the target selection spectrum, eaFnCas12a was engineered through site-directed mutagenesis. eaFnCas12a and one engineered variant (eaFnCas12a-RR), used for correcting human RS1 mutation responsible for X-linked retinoschisis, had a 3.28- to 4.04-fold improved activity compared with WT. Collectively, eaFnCas12a and its engineered variants can be used for genome-editing applications that requires high activity. Clustered regularly interspaced short palindromic repeat–Cas12a has been harnessed to manipulate the human genome; however, low cleavage efficiency and stringent protospacer adjacent motif hinder the use of Cas12a-based therapy and applications. Here, we have described a directional evolving and screening system in human cells to identify novel FnCas12a variants with high activity. By using this system, we identified IV-79 (enhanced activity FnCas12a, eaFnCas12a), which possessed higher DNA cleavage activity than WT FnCas12a. Furthermore, to widen the target selection spectrum, eaFnCas12a was engineered through site-directed mutagenesis. eaFnCas12a and one engineered variant (eaFnCas12a-RR), used for correcting human RS1 mutation responsible for X-linked retinoschisis, had a 3.28- to 4.04-fold improved activity compared with WT. Collectively, eaFnCas12a and its engineered variants can be used for genome-editing applications that requires high activity. Clustered regularly interspaced short palindromic repeat (CRISPR)–Cas systems, developed from the adaptive immune system of prokaryotes, has become a powerful genome manipulation tool in biological research and has great potential for gene therapy (1Chen X. Gonçalves M.A.F.V. DNA, RNA, and protein tools for editing the genetic information in human cells.iScience. 2018; 6: 247-263Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 2Komor A.C. Badran A.H. Liu D.R. CRISPR-based technologies for the manipulation of eukaryotic genomes.Cell. 2017; 168: 20-36Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar). Recently, Cas12a (also known as Cpf1), members of class 2 type V CRISPR–Cas family of nucleases, was reported to cleave the mammalian genome in human cells with distinctive characteristics, compared with the most commonly used SpCas9 (Streptococcus pyogenes Cas9) nuclease (3Zetsche B. Gootenberg J.S. Abudayyeh O.O. Slaymaker I.M. Makarova K.S. 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Cell Biol. 2020; 21: 661-677Crossref PubMed Scopus (63) Google Scholar). Subsequent structural studies revealed the molecular mechanisms of recognition and cleavage of DNA (18Dong D. Ren K. Qiu X. Zheng J. Guo M. Guan X. Liu H. Li N. Zhang B. Yang D. Ma C. Wang S. Wu D. Ma Y. Fan S. et al.The crystal structure of Cpf1 in complex with CRISPR RNA.Nature. 2016; 532: 522-526Crossref PubMed Scopus (164) Google Scholar, 19Yamano T. Nishimasu H. Zetsche B. Hirano H. Slaymaker I.M. Li Y. Fedorova I. Nakane T. Makarova K.S. Koonin E.V. Ishitani R. Zhang F. Nureki O. Crystal structure of Cpf1 in complex with guide RNA and target DNA.Cell. 2016; 165: 949-962Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). However, for Cas12a-based clinical and basic applications, several problems, including increasing its cleavage efficacy or fidelity and recognizing more flexible PAMs for target selection, exist (20Tu M. Lin L. Cheng Y. He X. Sun H. Xie H. Fu J. Liu C. Li J. Chen D. Xi H. Xue D. Liu Q. Zhao J. Gao C. et al.A 'new lease of life': FnCpf1 possesses DNA cleavage activity for genome editing in human cells.Nucleic Acids Res. 2017; 45: 11295-11304Crossref PubMed Scopus (61) Google Scholar). To address flexible PAM specificities, AsCas12a mutants (RR variant [S542R/K607R] and RVR variants [S542R/K548V/N552R]) have been identified, which recognize 5′-TYCV-3′ (Y: T/C; V: A/C/G) and 5′-TATV-3′ as PAM, respectively (21Gao L. Cox D.B.T. Yan W.X. Manteiga J.C. Schneider M.W. Yamano T. Nishimasu H. Nureki O. Crosetto N. Zhang F. Engineered Cpf1 variants with altered PAM specificities.Nat. Biotechnol. 2017; 35: 789-792Crossref PubMed Scopus (181) Google Scholar). A study demonstrated that these mutations could be used for engineering LbCas12a, FnCas12a, and MbCas12a (22Toth E. Czene B.C. Kulcsar P.I. Krausz S.L. Talas A. Nyeste A. Varga E. Huszar K. Weinhardt N. Ligeti Z. Borsy A.E. Fodor E. Welker E. Mb- and FnCpf1 nucleases are active in mammalian cells: Activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants.Nucleic Acids Res. 2018; 46: 10272-10285PubMed Google Scholar). Specifically, the RR variant recognizes 5′-TWTV-3′ (W: A/T, V: A/C/G) as PAM, whereas 5′-TYYV-3′ (Y: C/T, V: A/C/G) serves as the PAM for the RVR variant (22Toth E. Czene B.C. Kulcsar P.I. Krausz S.L. Talas A. Nyeste A. Varga E. Huszar K. Weinhardt N. Ligeti Z. Borsy A.E. Fodor E. Welker E. Mb- and FnCpf1 nucleases are active in mammalian cells: Activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants.Nucleic Acids Res. 2018; 46: 10272-10285PubMed Google Scholar). FnCas12a recognizes 5′-KYTV-3′ (K: T/G; Y: C/T; V: A/C/G) as PAM in human cells (20Tu M. Lin L. Cheng Y. He X. Sun H. Xie H. Fu J. Liu C. Li J. Chen D. Xi H. Xue D. Liu Q. Zhao J. Gao C. et al.A 'new lease of life': FnCpf1 possesses DNA cleavage activity for genome editing in human cells.Nucleic Acids Res. 2017; 45: 11295-11304Crossref PubMed Scopus (61) Google Scholar), which may be developed as tool for genome compared with AsCas12a and LbCas12a as PAM, to FnCas12a has low activity or activity target (20Tu M. Lin L. Cheng Y. He X. Sun H. Xie H. Fu J. Liu C. Li J. Chen D. Xi H. Xue D. Liu Q. Zhao J. Gao C. et al.A 'new lease of life': FnCpf1 possesses DNA cleavage activity for genome editing in human cells.Nucleic Acids Res. 2017; 45: 11295-11304Crossref PubMed Scopus (61) Google Scholar). engineering of the of FnCas12a may the genome editing activity. Here, we to and identify FnCas12a variants with high activity. on (20Tu M. Lin L. Cheng Y. He X. Sun H. Xie H. Fu J. Liu C. Li J. Chen D. Xi H. Xue D. Liu Q. Zhao J. Gao C. et al.A 'new lease of life': FnCpf1 possesses DNA cleavage activity for genome editing in human cells.Nucleic Acids Res. 2017; 45: 11295-11304Crossref PubMed Scopus (61) Google Scholar), we the and with FnCas12a in cells cells the A (also with on the of target in and (also with The revealed that we or mutations in A and the human system may be to the activity of FnCas12a variants through we variant of FnCas12a by mutations namely and of FnCas12a through Four and that could the namely and we mutagenesis of and that with we could 2 to mutations in the endonuclease recognition of and on this and we to the genetic information of The revealed that the mutations the we of FnCas12a variants of FnCas12a variants in cells and FnCas12a variants with high cleavage activity and Specifically, the of FnCas12a variants crRNA the and cleavage activity was that variants of of and of or higher activity compared with WT FnCas12a. we to the of the crRNA and to the of the variants and that crRNA had activity to high of this the of the crRNA distal to PAM for WT FnCas12a A and we crRNA to of the the we which had more than compared with WT FnCas12a with or to revealed namely and with activity as WT and higher fidelity more than activity of WT FnCas12a with than WT on A and revealed namely and with higher however, variants a in fidelity with mutants with higher activities are more to be To the activity of FnCas12a, we these variants as for with and variants we their activity in system and we variants from has higher activity than WT FnCas12a through crRNA and identified variants more than activity of WT FnCas12a A and However, on by with or to revealed that only variant had higher activity than WT. this variant to we that the IV-79 variant had activity and and activity FnCas12a The mutants can be to identify To the editing efficiency of eaFnCas12a with the Cas12a family and variants FnCas12a, and LbCas12a and AsCas12a variants and AsCas12a variants with we the protein and The that eaFnCas12a possesses higher activity than the Cas12a family and variants Specifically, eaFnCas12a possesses higher activity and than the four Cas12a family and variants and in human cells To target with 5′-KYTV-3′ (K: Y: C/T, V: A/C/G) PAM in and observed compared with eaFnCas12a higher activity and eaFnCas12a activity higher than WT FnCas12a to with (Y: C/T, V: A/C/G) PAM and and that eaFnCas12a possessed higher activity for genome To by was harnessed to the PAM in human cells L. Yang F. He X. Xie H. Liu X. Fu J. Xi H. X. Liu C. Z. J. Zhao J. F. cleavage PAM preferences of CRISPR-Cas in human 8: PubMed Scopus Google Scholar). of the from as The that the PAM for eaFnCas12a is (Y: C/T, V: also observed that the PAM of eaFnCas12a is more flexible than WT for target eaFnCas12a as as WT has its PAM for the recognition is To DNA cleavage activity of eaFnCas12a in human 18 genomic (Y: C/T, V: A/C/G) PAM for the with eaFnCas12a more activity with higher A and in and for and the eaFnCas12a variant higher To these we from the 18 genomic as for revealed efficacy of genome editing most editing A and which is with the from the the of sites, the are Furthermore, we off-target using and with in target effects of eaFnCas12a was observed demonstrated that the fidelity of eaFnCas12a was to that of FnCas12a. eaFnCas12a is engineered Cas12a nuclease with higher activity. Recently, was reported that RVR mutants and RR mutants PAM preferences with 5′-TATV-3′ A/C/G) PAM and 5′-TYCV-3′ PAM (22Toth E. Czene B.C. Kulcsar P.I. Krausz S.L. Talas A. Nyeste A. Varga E. Huszar K. Weinhardt N. Ligeti Z. Borsy A.E. Fodor E. Welker E. Mb- and FnCpf1 nucleases are active in mammalian cells: Activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants.Nucleic Acids Res. 2018; 46: 10272-10285PubMed Google Scholar). that these mutations may target selection of we the RVR and RR mutants of eaFnCas12a and and compared their PAM preferences and activities on target in with that in WT. with we observed that and PAM selection, compared with WT Specifically, WT and eaFnCas12a demonstrated activity with PAM, whereas activity and higher activity than 17 target with PAM and which used in PAM preferences and activity of observed that both eaFnCas12a and higher activity than WT or respectively we activity of target with A/C/G) PAM and and that eaFnCas12a had higher efficacy than WT and had the and we that the mutation with or may its target X-linked is the most of dystrophy in with L. R. in X-linked PubMed Scopus Google Scholar). Genome in gene correction great for its clinical To gene correction by we used the which the of RS1 the mutation from a with X-linked D. Xu T. M. Xu J. C. Cheng L. Yang R. Yang T. Zheng He X. R. X. Li J. Z. Zhao J. et X-linked in by novel Mol. 2017; PubMed Scopus Google Scholar). this system, is or WT DNA as is by Cas12a or 2 and with PAMs by and 2 A/C/G) and was the eaFnCas12a had the with or the observed a 3.28- and 4.04-fold in activity of the variant and compared with WT. we demonstrated that gene correction efficacy can be with eaFnCas12a and that one acid from to the for the of only a single mutants from and that may be a novel mutation the mutagenesis mutation of eaFnCas12a was from the mutagenesis. that the mutation was the of and To the improved activity of eaFnCas12a by we the crystal structure of FnCas12a in complex with crRNA and target DNA the of the which in PAM recognition with and the of to the of DNA, and the of and through which activity. However, that the a of of and of using the revealed that possessed the we that the may the and of FnCas12a to FnCas12a and DNA we the effects of of on activity was to or a in activity was observed of the of these to structural of FnCas12a and its activity. possessed activity and compared with WT FnCas12a, which may be of the of studies be to the mechanisms by which eaFnCas12a high activity. on the structure of FnCas12a S. P. G. of the Cpf1 endonuclease complex target DNA 2017; PubMed Scopus Google Scholar), we to identify mutants with higher activity. we and namely and However, these mutants for compared with eaFnCas12a we mutants in to these however, in activity was observed which the of effects of with may a in activity. to the Cas12a activity can be through crRNA engineering S. Lee J.M. Kang J.G. Lee Kim Kim S.H. K. Kim D. Kim Y.S. efficient genome editing by CRISPR-Cpf1 using CRISPR RNA with a Commun. 2018; PubMed Scopus (50) Google Scholar). engineered the crRNA and could the activity of eaFnCas12a target revealed compared with eaFnCas12a has higher editing efficacy with the engineered crRNA in of FnCas12a and engineered crRNA was The is a nuclease family with low off-target effects and PAMs compared with the the nucleases AsCas12a and LbCas12a are commonly used for gene FnCas12a can be used in and W.Y. Lebbink J.H.G. Kanaar R. Geijsen N. van der Oost J. Genome editing by natural and engineered CRISPR-associated nucleases.Nat. Chem. Biol. 2018; 14: 642-651Crossref PubMed Scopus (50) Google Scholar, P. F. H. plant and with and 2018; PubMed Scopus Google Scholar). However, FnCas12a has low cleavage activity (20Tu M. Lin L. Cheng Y. He X. Sun H. Xie H. Fu J. Liu C. Li J. Chen D. Xi H. Xue D. Liu Q. Zhao J. 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Lee K. of the crRNA Cpf1 gene editing in vitro and in Commun. 2018; PubMed Scopus Google Scholar). The mutagenesis screening has been to expand and fidelity of AsCas12a and LbCas12a (21Gao L. Cox D.B.T. Yan W.X. Manteiga J.C. Schneider M.W. Yamano T. Nishimasu H. Nureki O. Crosetto N. Zhang F. Engineered Cpf1 variants with altered PAM specificities.Nat. Biotechnol. 2017; 35: 789-792Crossref PubMed Scopus (181) Google Scholar, A.A. K. J. I. L. Joung J.K. Engineered CRISPR-Cas12a variants with activities and improved for and editing.Nat. Biotechnol. PubMed Scopus Google Scholar). the mutagenesis is in which the screening However, directional of Cas12a with mutagenesis to identify with in human cells mutagenesis is in of structural and The of this of to identify which may expand screening and for the target H. X. Yang F. Wang B. Li S. J. X. Cheng C. Z. Liu C. Zhao J. Zhang Y. Wu J. Gao C. Zhang J. et identified by directional screening in human Biol. 2020; PubMed Scopus Google Scholar). this we identified a novel FnCas12a variant with higher activity than WT FnCas12a through directional To the of we RR or RVR mutants to eaFnCas12a or than the variant both on and A/C/G) eaFnCas12a the of on PAMs also the RVR variant that the mutation is with RR or RVR To we which a the and the variant demonstrated the of with RR or RVR could be used for LbCas12a, or MbCas12a acid from these four nucleases and that is which the in AsCas12a or LbCas12a and the in analysis that to its a with may be for engineering AsCas12a or MbCas12a and Recently, editing was T-rich PAM with high the of PAM A.A. K. J. I. L. Joung J.K. Engineered CRISPR-Cas12a variants with activities and improved for and editing.Nat. Biotechnol. PubMed Scopus Google Scholar). editing is more genome whereas target selection may a for application J.R. A.A. J.M. Chen C. A. Liu D.R. genome editing breaks or PubMed Scopus Google Scholar, Q. Y. Xue C. Wang S. S. Z. Wang Y. A. Liu D.R. Gao C. genome editing in and Biotechnol. 2020; PubMed Scopus Google Scholar). we the of the eaFnCas12a for and may be used for genome editing we identified a through directional in human eaFnCas12a and its engineered variants could be used for genome editing applications that high activity with more flexible PAM for the of FnCas12a from The crRNA was by of DNA The and gene target used are in The used for FnCas12a mutations are in and The used for crRNA are in and DNA and genomic DNA by DNA the of the in the cells from and in in cells described (20Tu M. Lin L. Cheng Y. He X. Sun H. Xie H. Fu J. Liu C. Li J. Chen D. Xi H. Xue D. Liu Q. Zhao J. Gao C. et al.A 'new lease of life': FnCpf1 possesses DNA cleavage activity for genome editing in human cells.Nucleic Acids Res. 2017; 45: 11295-11304Crossref PubMed Scopus (61) Google Scholar, Y. X. Yang F. Zhang L. Zheng J. X. J. F. of PAMs for DNA cleavage in human PubMed Scopus Google Scholar). cells RS1 by with and selection with cells in had single To RS1 the for The information has been described He Liu Sun Zhang J.S. Zhao J. Liu F. by in human 2018; 45: Scopus Google Scholar). The FnCas12a by mutagenesis by Specifically, FnCas12a was with or The or by mutagenesis and with the the The from The from single and double mutants and to the structure of FnCas12a S. P. G. of the Cpf1 endonuclease complex target DNA 2017; PubMed Scopus Google Scholar). The was described on in and with Cas12a and crRNA by the on was to the cells on or for or genomic DNA on in with 2 and to a of and to to with for 2 and was on was by the a is the of the and are the of the cleavage to the using of in and of the to using the and from the using to the using with this in this The used for and are in in on and with FnCas12a, crRNA and 2 WT DNA by on was on for on to as by or The for was The that the of this study are in the The are the are The that have of with the of this Li and Sun for and from Wang of of the C. J. X. Y. H. and X. L. the and B. L. M. C. J. Y. and F. G. and F. G. the have and the was by from of of and and of of of research for of Lin of and and and of of and

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