A Tale of Two Moieties: Rapidly Evolving CRISPR/Cas-Based Genome Editing
2020; Elsevier BV; Volume: 45; Issue: 10 Linguagem: Inglês
10.1016/j.tibs.2020.06.003
ISSN1362-4326
Autores Tópico(s)Innovation and Socioeconomic Development
ResumoCRISPR/Cas with different modules for independent target binding and cleavage has evolved to achieve convenient and precise genome editing.The endonuclease effector in conventional CRISPR/Cas genome editing systems can be replaced by nucleobase deaminases and the resulting base editors (BEs) enable single base changes.By fusing CRISPR/Cas with reverse transcriptases, prime editors (PEs) represent a new way to accomplish genetic changes, including all types of base substitutions, small indels, and their combinations.Both BEs and PEs are of potential in correcting disease-associated mutations.Genome-wide and/or transcriptome-wide off-target mutations are catalyzed by the nucleobase deaminase effector in BEs, which are independent of the fused gRNA/Cas moiety. Two major moieties in genome editing are required for precise genetic changes: the locator moiety for target binding and the effector moiety for genetic engineering. By taking advantage of CRISPR/Cas, which consists of different modules for independent target binding and cleavage, a spectrum of precise and versatile genome editing technologies have been developed for broad applications in biomedical research, biotechnology, and therapeutics. Here, we briefly summarize the progress of genome editing systems from a view of both locator and effector moieties and highlight the advance of newly reported CRISPR-conjugated base editing and prime editing systems. We also underscore distinct mechanisms of off-target effects in CRISPR-conjugated systems and further discuss possible strategies to reduce off-target mutations in the future. Two major moieties in genome editing are required for precise genetic changes: the locator moiety for target binding and the effector moiety for genetic engineering. By taking advantage of CRISPR/Cas, which consists of different modules for independent target binding and cleavage, a spectrum of precise and versatile genome editing technologies have been developed for broad applications in biomedical research, biotechnology, and therapeutics. Here, we briefly summarize the progress of genome editing systems from a view of both locator and effector moieties and highlight the advance of newly reported CRISPR-conjugated base editing and prime editing systems. We also underscore distinct mechanisms of off-target effects in CRISPR-conjugated systems and further discuss possible strategies to reduce off-target mutations in the future. The completion of human genome project in the beginning of this century [1.Lander E.S. et al.Initial sequencing and analysis of the human genome.Nature. 2001; 409: 860-921Crossref PubMed Scopus (15711) Google Scholar,2.Venter J.C. et al.The sequence of the human genome.Science. 2001; 291: 1304-1351Crossref PubMed Scopus (9623) Google Scholar] and the application of affordable high-throughput sequencing technologies in the past decade [3.Metzker M.L. Sequencing technologies - the next generation.Nat. Rev. Genet. 2010; 11: 31-46Crossref PubMed Scopus (4365) Google Scholar] have led life science researches to the post-genome era with genome-wide understanding of functional genomic elements related to human health and diseases. Importantly, the advent of practical genome editing technologies provides powerful methods to change genetic information, which benefits not only basic research aiming to decipher how different genotypes result in distinct phenotypes but also preclinical study to cure human diseases caused by genetic mutations. To target any genomic locus for desired DNA changes, two major moieties, a locator (see Glossary) and an effector, are usually required for competent genome editing. The locator moiety is designed to recognize and bind to a specific genomic locus, which guides the effector moiety for subsequent change of DNA sequence. In last two decades or so, programmable genome editing systems have been mainly evolved from fusions of endonucleases to locators, such as zinc finger (ZF) motifs [4.Beerli R.R. Barbas III, C.F. Engineering polydactyl zinc-finger transcription factors.Nat. Biotechnol. 2002; 20: 135-141Crossref PubMed Scopus (352) Google Scholar] and transcription activator-like effector (TALE) repeats [5.Boch J. Bonas U. Xanthomonas AvrBs3 family-type III effectors: discovery and function.Annu. Rev. Phytopathol. 2010; 48: 419-436Crossref PubMed Scopus (554) Google Scholar] (Box 1), to the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based technologies [6.Hsu P.D. et al.Development and applications of CRISPR-Cas9 for genome engineering.Cell. 2014; 157: 1262-1278Abstract Full Text Full Text PDF PubMed Scopus (2517) Google Scholar, 7.Wright A.V. et al.Biology and applications of CRISPR systems: harnessing nature's toolbox for genome engineering.Cell. 2016; 164: 29-44Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 8.Komor A.C. et al.CRISPR-based technologies for the manipulation of eukaryotic genomes.Cell. 2017; 168: 20-36Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar]. Unlike ZFs and TALEs, which are fused with a heterogeneous FokI endonuclease for genome editing (Box 1), CRISPR/Cas proteins are featured by their dual functions. In addition to their DNA/RNA binding activity together with gRNA, CRISPR/Cas proteins can also process DNA/RNA cleavage activity with their endonuclease domains [9.Jinek M. et al.Structures of Cas9 endonucleases reveal RNA-mediated conformational activation.Science. 2014; 343: 1247997Crossref PubMed Scopus (491) Google Scholar, 10.Ran F.A. et al.In vivo genome editing using Staphylococcus aureus Cas9.Nature. 2015; 520: 186-191Crossref PubMed Scopus (1164) Google Scholar, 11.Zetsche B. et al.Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.Cell. 2015; 163: 759-771Abstract Full Text Full Text PDF PubMed Google Scholar, 12.Abudayyeh O.O. et al.C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector.Science. 2016; 353aaf5573Crossref PubMed Scopus (519) Google Scholar]. This makes CRISPR/Cas a convenient method for genome editing. Indeed, since it appeared in the early 2010s [11.Zetsche B. et al.Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.Cell. 2015; 163: 759-771Abstract Full Text Full Text PDF PubMed Google Scholar,13.Cong L. et al.Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (7045) Google Scholar, 14.Jinek M. et al.RNA-programmed genome editing in human cells.Elife. 2013; 2e00471Crossref PubMed Scopus (1139) Google Scholar, 15.Mali P. et al.RNA-guided human genome engineering via Cas9.Science. 2013; 339: 823-826Crossref PubMed Scopus (4829) Google Scholar], CRISPR/Cas has been widely applied in genome editing of both single gene study and genome-wide screening, from bacteria to mammals [6.Hsu P.D. et al.Development and applications of CRISPR-Cas9 for genome engineering.Cell. 2014; 157: 1262-1278Abstract Full Text Full Text PDF PubMed Scopus (2517) Google Scholar, 7.Wright A.V. et al.Biology and applications of CRISPR systems: harnessing nature's toolbox for genome engineering.Cell. 2016; 164: 29-44Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 8.Komor A.C. et al.CRISPR-based technologies for the manipulation of eukaryotic genomes.Cell. 2017; 168: 20-36Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar]. However, although revolutionary, CRISPR/Cas systems were not always precise, but with unwanted side-products; there has been an aim to have improved precision in the application of genome editing to treat genetic diseases associated with single base mutations. Recently, by fusing CRISPR/Cas proteins (as the genome locator) with different types of effector moieties, such as nucleobase deaminases [16.Komor A.C. et al.Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature. 2016; 533: 420-424Crossref PubMed Scopus (1103) Google Scholar,17.Gaudelli N.M. et al.Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage.Nature. 2017; 551: 464-471Crossref PubMed Scopus (697) Google Scholar] or reverse transcriptases [18.Anzalone A.V. et al.Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (243) Google Scholar], more precise and versatile genome editing technologies have been developed to achieve single nucleotide editing at target sites. In this review, we discuss the evolution of genome editing technologies in terms of two moieties, emphasize newly reported base editing and prime editing technologies based on CRISPR/Cas systems that have increased precision in gene editing, and further dissect underlined mechanisms that may account for their unwanted off-target (OT) effects for future improvement. (See Table 1).Box 1Genome Targeting Achieved by Protein LocatorsTo target any specific genomic site is one of the primary requirements for a programmable genome editing technology. Site-specific nucleases have long been applied in DNA recombination in vitro and therefore were first thought to be used for gene editing. For example, meganucleases, a type of endonucleases that recognize long DNA sequences (~12–40 bp), have been applied and engineered to generate DSBs at specific loci in genomic DNA [141.Rouet P. et al.Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease.Mol. Cell. Biol. 1994; 14: 8096-8106Crossref PubMed Google Scholar,142.Epinat J.C. et al.A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells.Nucleic Acids Res. 2003; 31: 2952-2962Crossref PubMed Scopus (166) Google Scholar]. However, due to their limited recognition sites and the difficulty to program their targeting specificities, meganucleases were not suitable in certain applications, such as in high-throughput screening assays.The first applicable locator for genome editing was developed with ZF motifs, originally discovered in transcription factors in Xenopus laevis [143.Miller J. et al.Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes.EMBO J. 1985; 4: 1609-1614Crossref PubMed Google Scholar,144.Klug A. The discovery of zinc fingers and their applications in gene regulation and genome manipulation.Annu. Rev. Biochem. 2010; 79: 213-231Crossref PubMed Scopus (413) Google Scholar]. By fusing an array of ZF motifs as the locator with the cleavage domain of FokI endonuclease as the effector, ZF nucleases (ZFNs) were developed to fulfill genome editing [145.Kim Y.G. et al.Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1156-1160Crossref PubMed Scopus (1089) Google Scholar], theoretically at any given genomic locus. The specificity of ZFNs is rendered by the customized array of ZF motifs, each of which consists of about 30 amino acids to recognize a definite nucleotide triplet [146.Pavletich N.P. Pabo C.O. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A.Science. 1991; 252: 809-817Crossref PubMed Scopus (1642) Google Scholar,147.Rebar E.J. Pabo C.O. Zinc finger phage: affinity selection of fingers with new DNA-binding specificities.Science. 1994; 263: 671-673Crossref PubMed Google Scholar]. Within a designed ZFN, different ZF motifs can be combined to recognize ~9–18 bp at the targeted genomic locus for subsequent editing [148.Bibikova M. et al.Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases.Genetics. 2002; 161: 1169-1175Crossref PubMed Google Scholar]. However, the application of ZFNs at most genomic target sites has remained challenging due to the crosstalk between adjacent ZF motifs that interferes with their binding to the corresponding DNA.The ZFN-based technology was the only programmable method to engineer genomic DNA sequences for a while, prior to the appearance of TALE nucleases (TALENs) in 2011 [149.Miller J.C. et al.A TALE nuclease architecture for efficient genome editing.Nat. Biotechnol. 2011; 29: 143-148Crossref PubMed Scopus (1323) Google Scholar]. The TALEN system uses TALE repeats, from a bacterial plant pathogen Xanthomonas, as the locator [5.Boch J. Bonas U. Xanthomonas AvrBs3 family-type III effectors: discovery and function.Annu. Rev. Phytopathol. 2010; 48: 419-436Crossref PubMed Scopus (554) Google Scholar]. Each TALE repeat composes of 33–35 amino acids to distinguish a single base pair of DNA [150.Boch J. et al.Breaking the code of DNA binding specificity of TAL-type III effectors.Science. 2009; 326: 1509-1512Crossref PubMed Scopus (1580) Google Scholar,151.Moscou M.J. Bogdanove A.J. A simple cipher governs DNA recognition by TAL effectors.Science. 2009; 326: 1501Crossref PubMed Scopus (1240) Google Scholar]; this leads to increased flexibility in designing customized TALENs to engineer most genetic loci by combining matched TALE repeats. By fusing an array of TALE DNA binding domains that recognize designated base pairs to the cleavage domain of FokI endonuclease, the fusion protein can bind to a specific DNA sequence without the interference of each TALE domain in the array [149.Miller J.C. et al.A TALE nuclease architecture for efficient genome editing.Nat. Biotechnol. 2011; 29: 143-148Crossref PubMed Scopus (1323) Google Scholar,152.Cermak T. et al.Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting.Nucleic Acids Res. 2011; 39e82Crossref PubMed Scopus (1339) Google Scholar,153.Christian M. et al.Targeting DNA double-strand breaks with TAL effector nucleases.Genetics. 2010; 186: 757-761Crossref PubMed Scopus (1029) Google Scholar]. Nonetheless, the construction of TALEN vectors is complicated due to the homologous recombination of repetitive DNA sequences to express TALE repeats.Table 1Representative Genome EditorsGenome editorLocatorEffectorPAMLocator-dependent OT effectsLocator-independent OT effectsRefsZFNZF motifFokI nuclease–+++–[145.Kim Y.G. et al.Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1156-1160Crossref PubMed Scopus (1089) Google Scholar,148.Bibikova M. et al.Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases.Genetics. 2002; 161: 1169-1175Crossref PubMed Google Scholar]TALENTALE repeatFokI nuclease–+++–[149.Miller J.C. et al.A TALE nuclease architecture for efficient genome editing.Nat. Biotechnol. 2011; 29: 143-148Crossref PubMed Scopus (1323) Google Scholar,152.Cermak T. et al.Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting.Nucleic Acids Res. 2011; 39e82Crossref PubMed Scopus (1339) Google Scholar,153.Christian M. et al.Targeting DNA double-strand breaks with TAL effector nucleases.Genetics. 2010; 186: 757-761Crossref PubMed Scopus (1029) Google Scholar]Cas9Cas9/gRNACas9 nucleaseNGG+++–[13.Cong L. et al.Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (7045) Google Scholar,14.Jinek M. et al.RNA-programmed genome editing in human cells.Elife. 2013; 2e00471Crossref PubMed Scopus (1139) Google Scholar,154.Mali P. et al.CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.Nat. Biotechnol. 2013; 31: 833-838Crossref PubMed Scopus (1050) Google Scholar]Cas12aCas12a/gRNACas12a nucleaseTTTV++–[11.Zetsche B. et al.Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.Cell. 2015; 163: 759-771Abstract Full Text Full Text PDF PubMed Google Scholar]Cas9-VQRCas9-VQR/gRNACas9 nucleaseNGA+++–[29.Kleinstiver B.P. et al.Engineered CRISPR-Cas9 nucleases with altered PAM specificities.Nature. 2015; 523: 481-485Crossref PubMed Scopus (724) Google Scholar]xCas9xCas9/gRNACas9 nucleaseNG, GAA, GAT++–[30.Hu J.H. et al.Evolved Cas9 variants with broad PAM compatibility and high DNA specificity.Nature. 2018; 556: 57-63Crossref PubMed Scopus (431) Google Scholar]Cas9-NGCas9-NG/gRNACas9 nucleaseNG+++–[31.Nishimasu H. et al.Engineered CRISPR-Cas9 nuclease with expanded targeting space.Science. 2018; 361: 1259-1262Crossref PubMed Scopus (195) Google Scholar]Cas9-NRRHCas9-NRRH/gRNACas9 nucleaseNRRH+++–[32.Miller S.M. et al.Continuous evolution of SpCas9 variants compatible with non-G PAMs.Nat. Biotechnol. 2020; 38: 471-481Crossref PubMed Scopus (19) Google Scholar]Cas13aCas13a/gRNACas13a nucleaseH+–[34.Abudayyeh O.O. et al.RNA targeting with CRISPR-Cas13.Nature. 2017; 550: 280-284Crossref PubMed Scopus (373) Google Scholar]nCas9nCas9/gRNA pairnCas9NGG++–[108.Ran F.A. et al.Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.Cell. 2013; 154: 1380-1389Abstract Full Text Full Text PDF PubMed Scopus (1792) Google Scholar,109.Shen B. et al.Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects.Nat. Methods. 2014; 11: 399-402Crossref PubMed Scopus (452) Google Scholar]dCas9-FokIdCas9/gRNA pairFokI nucleaseNGG+–[110.Tsai S.Q. et al.Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing.Nat. Biotechnol. 2014; 32: 569-576Crossref PubMed Scopus (581) Google Scholar,155.Guilinger J.P. et al.Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification.Nat. Biotechnol. 2014; 32: 577-582Crossref PubMed Scopus (491) Google Scholar]eSpCas9eSpCas9/gRNACas9 nucleaseNGG––[125.Slaymaker I.M. et al.Rationally engineered Cas9 nucleases with improved specificity.Science. 2016; 351: 84-88Crossref PubMed Scopus (998) Google Scholar]SpCas9-HFSpCas9-HF/gRNACas9 nucleaseNGG––[126.Kleinstiver B.P. et al.High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.Nature. 2016; 529: 490-495Crossref PubMed Scopus (1045) Google Scholar]HypaCas9HypaCas9/gRNACas9 nucleaseNGG––[127.Chen J.S. et al.Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.Nature. 2017; 550: 407-410Crossref PubMed Scopus (330) Google Scholar]Sniper-Cas9Sniper-Cas9/gRNACas9 nucleaseNGG––[128.Lee J.K. et al.Directed evolution of CRISPR-Cas9 to increase its specificity.Nat. Commun. 2018; 9: 3048Crossref PubMed Scopus (89) Google Scholar]BE3nCas9/gRNArA1NGG+++DNA: +++, RNA: +++[16.Komor A.C. et al.Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature. 2016; 533: 420-424Crossref PubMed Scopus (1103) Google Scholar]YE1-BE3nCas9/gRNArA1-YE1NGG+++DNA: –, RNA: –[68.Kim Y.B. et al.Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions.Nat. Biotechnol. 2017; 35: 371-376Crossref PubMed Scopus (258) Google Scholar]YEE-BE3nCas9/gRNArA1-YEENGG+++DNA: –, RNA: –[68.Kim Y.B. et al.Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions.Nat. Biotechnol. 2017; 35: 371-376Crossref PubMed Scopus (258) Google Scholar]BE4nCas9/gRNArA1NGG+++DNA: +++, RNA: +++[72.Komor A.C. et al.Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity.Sci. Adv. 2017; 3eaao4774Crossref PubMed Scopus (350) Google Scholar]eBEnCas9/gRNArA1NGG+++DNA: +++, RNA: +++[73.Wang L. et al.Enhanced base editing by co-expression of free uracil DNA glycosylase inhibitor.Cell Res. 2017; 27: 1289-1292Crossref PubMed Scopus (42) Google Scholar]hA3A-BE3nCas9/gRNAhA3ANGG+++DNA: +++, RNA: +++[63.Wang X. et al.Efficient base editing in methylated regions with a human APOBEC3A-Cas9 fusion.Nat. Biotechnol. 2018; 36: 946-949Crossref PubMed Scopus (59) Google Scholar]hA3A-BE3-Y130FnCas9/gRNAhA3A-Y130FNGG+++DNA: +++, RNA: –[63.Wang X. et al.Efficient base editing in methylated regions with a human APOBEC3A-Cas9 fusion.Nat. Biotechnol. 2018; 36: 946-949Crossref PubMed Scopus (59) Google Scholar]hA3A-BE3-Y132DnCas9/gRNAhA3A-Y132DNGG+++DNA: +++, RNA: +[63.Wang X. et al.Efficient base editing in methylated regions with a human APOBEC3A-Cas9 fusion.Nat. Biotechnol. 2018; 36: 946-949Crossref PubMed Scopus (59) Google Scholar]eA3A-BE3nCas9/gRNAA3A-N57QNGG++DNA: +++, RNA: +[69.Gehrke J.M. et al.An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities.Nat. Biotechnol. 2018; 36: 977-982Crossref PubMed Scopus (99) Google Scholar]SaKKH-BE3nSaKKHCas9/gRNArA1NNNRRT+++DNA: +++, RNA: +++[68.Kim Y.B. et al.Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions.Nat. Biotechnol. 2017; 35: 371-376Crossref PubMed Scopus (258) Google Scholar]Target-AIDnCas9/gRNASea lamprey CDANGG+++DNA: +++, RNA: –[51.Nishida K. et al.Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.Science. 2016; 353aaf8729Crossref PubMed Scopus (631) Google Scholar]dCas12a-BEdLbCas12a/gRNArA1TTTV++DNA: +++, RNA: +++[59.Li X. et al.Base editing with a Cpf1-cytidine deaminase fusion.Nat. Biotechnol. 2018; 36: 324-327Crossref PubMed Scopus (112) Google Scholar]BEACON1dLbCas12a/gRNAEngineered hA3ATTTV++DNA: +++, RNA: +[75.Wang X. et al.Cas12a base editors induce efficient and specific editing with low DNA damage response.Cell Rep. 2020; 31: 107723Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]BEACON2dLbCas12a/gRNAEngineered hA3ATTTV++DNA: +++, RNA: –[75.Wang X. et al.Cas12a base editors induce efficient and specific editing with low DNA damage response.Cell Rep. 2020; 31: 107723Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]enAsBEdenAsCas12a/gRNArA1VTTV, TTTT, TTCN/TATV+DNA: +++, RNA: +++[74.Kleinstiver B.P. et al.Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing.Nat. Biotechnol. 2019; 37: 276-282Crossref PubMed Scopus (68) Google Scholar]PBEnCas9/gRNArA1NGG+++DNA: +++, RNA: +++[61.Zong Y. et al.Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion.Nat. Biotechnol. 2017; 35: 438-440Crossref PubMed Scopus (280) Google Scholar]A3A-PBEnCas9/gRNAhA3ANGG+++DNA: +++, RNA: +++[64.Zong Y. et al.Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A.Nat. Biotechnol. 2018; 36: 950-953Crossref Scopus (68) Google Scholar]ABE7.10nCas9/gRNATadA-TadA*NGG+++DNA: –, RNA: +[17.Gaudelli N.M. et al.Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage.Nature. 2017; 551: 464-471Crossref PubMed Scopus (697) Google Scholar]ABE8enCas9/gRNATadA-TadA-8eNGG+++DNA: +++, RNA: +++[83.Richter M.F. et al.Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity.Nat. Biotechnol. 2020; (Published online March 15, 2020. https://doi.org/10.1038/s41587-020-0453-z)Google Scholar]ABE8e-V106WnCas9/gRNATadA-TadA-8e-V106WNGG+++DNA: +, RNA: ++[83.Richter M.F. et al.Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity.Nat. Biotechnol. 2020; (Published online March 15, 2020. https://doi.org/10.1038/s41587-020-0453-z)Google Scholar]LbABE8edLbCas12a/gRNATadA-TadA-8eTTTV++DNA: +++, RNA: +++[83.Richter M.F. et al.Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity.Nat. Biotechnol. 2020; (Published online March 15, 2020. https://doi.org/10.1038/s41587-020-0453-z)Google Scholar]STEME-1nCas9/gRNAhA3A-TadA-TadA*NGG+++DNA: +++, RNA: +++[83.Richter M.F. et al.Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity.Nat. Biotechnol. 2020; (Published online March 15, 2020. https://doi.org/10.1038/s41587-020-0453-z)Google Scholar]ABE-P1nCas9/gRNATadA-TadA*NGG+++DNA: –, RNA: +[92.Hua K. et al.Precise A.T to G.C base editing in the rice genome.Mol. Plant. 2018; 11: 627-630Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]ABE-P2nSaCas9/gRNATadA-TadA*NNGRRT+++DNA: –, RNA: +[92.Hua K. et al.Precise A.T to G.C base editing in the rice genome.Mol. Plant. 2018; 11: 627-630Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]rBE14nCas9/gRNATadA-TadA*NGG+++DNA: –, RNA: +[93.Yan F. et al.Highly efficient A.T to G.C base editing by Cas9n-guided tRNA adenosine deaminase in rice.Mol. Plant. 2018; 11: 631-634Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]PE1dCas9/gRNAM-MLV RTaseNGG+DNA: ?, RNA: ?[18.Anzalone A.V. et al.Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (243) Google Scholar]PE2nCas9/gRNAM-MLV RTaseNGG+DNA: ?, RNA: ?[18.Anzalone A.V. et al.Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (243) Google Scholar]PE3nCas9/gRNAM-MLV RTaseNGG+DNA: ?, RNA: ?[18.Anzalone A.V. et al.Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (243) Google Scholar]PPEnCas9/gRNAM-MLV RTaseNGG+DNA: ?, RNA: ?[94.Lin Q. et al.Prime genome editing in rice and wheat.Nat. Biotechnol. 2020; 38: 582-585Crossref PubMed Scopus (21) Google Scholar] Open table in a new tab To target any specific genomic site is one of the primary requirements for a programmable genome editing technology. Site-specific nucleases have long been applied in DNA recombination in vitro and therefore were first thought to be used for gene editing. For example, meganucleases, a type of endonucleases that recognize long DNA sequences (~12–40 bp), have been applied and engineered to generate DSBs at specific loci in genomic DNA [141.Rouet P. et al.Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease.Mol. Cell. Biol. 1994; 14: 8096-8106Crossref PubMed Google Scholar,142.Epinat J.C. et al.A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells.Nucleic Acids Res. 2003; 31: 2952-2962Crossref PubMed Scopus (166) Google Scholar]. However, due to their limited recognition sites and the difficulty to program their targeting specificities, meganucleases were not suitable in certain applications, such as in high-throughput screening assays. The first applicable locator for genome editing was developed with ZF motifs, originally discovered in transcription factors in Xenopus laevis [143.Miller J. et al.Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes.EMBO J. 1985; 4: 1609-1614Crossref PubMed Google Scholar,144.Klug A. The discovery of zinc fingers and their applications in gene regulation and genome manipulation.Annu. Rev. Biochem. 2010; 79: 213-231Crossref PubMed Scopus (413) Google Scholar]. By fusing an array of ZF motifs as the locator with the cleavage domain of FokI endonuclease as the effector, ZF nucleases (ZFNs) were developed to fulfill genome editing [145.Kim Y.G. et al.Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1156-1160Crossref PubMed Scopus (1089) Google Scholar], theoretically at any given genomic locus. The specificity of ZFNs is rendered by the customized array of ZF motifs, each of which consists of about 30 amino acids to recognize a definite nucleotide triplet [146.Pavletich N.P. Pabo C.O. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A.Science. 1991; 252: 809-817Crossref PubMed Scopus (1642) Google Scholar,147.Rebar E.J. Pabo C.O. Zinc finger phage: affinity selection of fingers with new DNA-binding specificities.Science. 1994; 263: 671-673Crossref PubMed Google Scholar]. Within a designed ZFN, different ZF motifs can be combined to recognize ~9–18 bp at the targeted genomic locus for subsequent editing [148.Bibikova M. et al.Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases.Genetics. 2002; 161: 1169-1175Crossref PubMed Google Scholar]. However, the application of ZFNs at most genomic target sites has remained challenging due to the crosstalk between adjacent ZF motifs that interferes with their binding to the corresponding DNA. The ZFN-based technology was the only programmable method to engineer genomic DNA sequences for a while, prior to the appearance of TALE nucleases (TALENs) in 2011 [149.Miller J.C. et al.A TALE nuclease architecture for efficient genome editing.Nat. Biotechnol. 2011; 29: 143-148Crossref PubMed Scopus (1323) Google Scholar]. The TALEN system uses TALE repeats, from a bacterial plant pathogen Xanthomonas, as the locator [5.Boch J. Bonas U. Xanthomonas AvrBs3 family-type III effectors: discovery and function.Annu. Rev. Phytopathol. 2010; 48: 419-436Crossref PubMed Scopus (554) Google Scholar]. Each TALE repeat composes of 33–35 amino acids to distinguish a single base pair of DNA [150.Boch J. et al.Breaking the code of DNA binding specificity of TAL-type III effectors.Science. 2009; 326: 1509-1512Crossref PubMed Scopus (1580) Google Scholar,151.Moscou M.J. Bogdanove A.J. A simple cipher governs DNA recognition by TAL effectors.Science. 2009; 326: 1501Crossref PubMed Scopus (1240) Google Scholar]; this leads to increased flexibility in designing customized TALENs to engineer most genetic loci by combining matched TALE repeats. By fusing an array of TALE DNA binding domains that recognize designated base pairs to the cleavage domain of FokI endonuclease, the fusion protein can bind to a specific DNA sequence without the interference of each TALE domain in the array [149.Miller J.C. et al.A TALE nuclease architecture for efficient genome editing.Nat. Biotechnol. 2011; 29: 143-148Crossref PubMed Scopus (1323) Google Scholar,152.Cermak T. et al.Efficient desig
Referência(s)