靶向DNA的Ⅱ类CRISPR/Cas系统的蛋白工程化改造

doi:10.12211/2096-8280.2022-040

合成生物学 ›› 2023, Vol. 4 ›› Issue (1): 86-101.DOI: 10.12211/2096-8280.2022-040

靶向DNA的Ⅱ类CRISPR/Cas系统的蛋白工程化改造

梁丽亚, 刘嵘明

大连理工大学生物工程学院，辽宁大连 116024

收稿日期:2022-07-18 修回日期:2022-09-28 出版日期:2023-02-28 发布日期:2023-03-07
通讯作者: 刘嵘明
作者简介:梁丽亚（1987—），女，博士，副教授。研究方向为：基因编辑、合成生物学、代谢工程。E-mail：liya_liang@dlut.edu.cn
刘嵘明（1985—），男，博士，教授。研究方向为：（1）高精度CRISPR基因编辑核酸酶的开发与应用；（2）高通量CRISPR基因组编辑工程策略开发与应用；（3）细胞工厂的功能预测与智能化设计；（4）构建细胞工厂合成生物基化学品、生物燃料以及高附加值产品。E-mail：rongming_liu@dlut.edu.cn
第一联系人：梁丽亚（1987—），女，博士，副教授。研究方向为：基因编辑、合成生物学、代谢工程。
基金资助:
国家自然科学基金(22278058);大连理工大学基本科研业务费（DUT22RC（3）012,DUT22RC（3）013）

Protein engineering of DNA targeting type Ⅱ CRISPR/Cas systems

Liya LIANG, Rongming LIU

School of Bioengineering，Dalian University of Technology，Dalian 116024，Liaoning，China

Received:2022-07-18 Revised:2022-09-28 Online:2023-02-28 Published:2023-03-07
Contact: Rongming LIU

摘要/Abstract

摘要：

Clustered regularly interspaced short palindromic repeats （CRISPR）/CRISPR-associated （Cas） protein是细菌或古细菌特有的一种获得性免疫系统，自从研究人员将其改造为基因编辑工具之后，CRISPR/Cas系统高效的基因编辑能力对生命科学、生物工程、生物医药、食品及农业科学等多个领域引发了革命性的影响。然而，研究者们发现CRISPR/Cas系统存在脱靶效应、PAM位点识别范围有限等问题，限制了CRISPR/Cas的应用。为了解决这些问题，针对Cas蛋白进行工程化改造已经成为了优化及开发CRISPR/Cas系统的重要策略。本文以Ⅱ类CRISPR/Cas系统中靶向DNA的CRISPR/Cas9以及CRISPR/Cas12a为例，聚焦近年来针对Cas9和Cas12a蛋白的优化改造方法及相关进展进行系统阐述总结，包括Cas蛋白的工程化改造提升基因编辑特异性及改变PAM识别能力，以CRISPR/Cas系统作为基因定位工具开发新功能，结合外源蛋白调控CRISPR/Cas系统功能。这些工作开发了系列高特异性、高精度的CRISPR/Cas系统，极大地扩展了CRISPR/Cas系统的功能及适用范围，为CRISPR/Cas系统的多领域应用做出了重要贡献。

关键词: CRISPR, Cas9, Cas12a, Cas 蛋白质工程, 基因编辑

Abstract:

With different types of nucleases, genome editing technologies have opened up the possibility for targeting and modifying specific gene sequences, which show potential applications in basic and applied aspects of biotechnology research. Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are artificial proteins generated by fusing a specific DNA-binding domain with a restriction enzyme FokI DNA-cleavage domain, which arise from their ability to customize the DNA-binding domain for recognition of targeting sequences and cleaving them by the FokI domain. However, the design and construction of such a system are time consuming, laborious and costly. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) protein is a unique acquired immune system of bacteria or archaea. Since researchers constructed the CRISPR/Cas system for gene editing, its high efficiency has revolutionized a variety of fields such as life sciences, bioengineering, biomedicine, food, and agricultural sciences. However, the CRISPR/Cas system still has some challenges, such as off-target effect and limited PAM site recognition range, which limit its further applications. In order to solve these problems, molecular engineering of Cas proteins has become an important strategy for developing and optimizing CRISPR/Cas systems. In this study, with CRISPR/Cas9 and CRISPR/Cas12a selected as representative examples for DNA-targeting Class II CRISPR/Cas systems, we focus on the optimization and modification methods, and progress of Cas9 and Cas12a proteins achieved within recent years, such as Cas protein engineering for improved on-target specificity and expanded PAM scopes, developing new functions using CRISPR/Cas systems as gene targeting tools, and introducing exogenous protein domains to regulate CRISPR/Cas functions. These studies have generated a series of high-specificity and high-precision CRISPR/Cas systems, which have greatly expanded their functions and scopes, and made important contributions to the wide-range applications of CRISPR/Cas systems. {L-End}

Key words: CRISPR, Cas9, Cas12a, Cas protein engineering, gene editing

中图分类号:

Q812

梁丽亚, 刘嵘明. 靶向DNA的Ⅱ类CRISPR/Cas系统的蛋白工程化改造[J]. 合成生物学, 2023, 4(1): 86-101.

Liya LIANG, Rongming LIU. Protein engineering of DNA targeting type Ⅱ CRISPR/Cas systems[J]. Synthetic Biology Journal, 2023, 4(1): 86-101.

图/表 6

参考文献 118

1	ZEKONYTE U, BACMAN S R, SMITH J, et al. Mitochondrial targeted meganuclease as a platform to eliminate mutant mtDNA in vivo [J]. Nature Communications, 2021, 12: 3210.
2	CARROLL D. Genome engineering with zinc-finger nucleases[J]. Genetics, 2011, 188(4): 773-782.
3	NEMUDRYI A A, VALETDINOVA K R, MEDVEDEV S P, et al. TALEN and CRISPR/Cas genome editing systems: tools of discovery[J]. Acta Naturae, 2014, 6(3): 19-40.
4	RAN F A, HSU P D, WRIGHT J, et al. Genome engineering using the CRISPR-Cas9 system[J]. Nature Protocols, 2013, 8(11): 2281-2308.
5	EPINAT J C, ARNOULD S, CHAMES P, et al. A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells[J]. Nucleic Acids Research, 2003, 31(11): 2952-2962.
6	GAJ T, GERSBACH C A, BARBARS C F III, et al. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering[J]. Trends in Biotechnology, 2013, 31(7): 397-405.
7	WANG H X, LI M Q, LEE C M, et al. CRISPR/Cas9-based genome editing for disease modeling and therapy: challenges and opportunities for nonviral delivery[J]. Chemical Reviews, 2017, 117(15): 9874-9906.
8	JIANG W Y, BIKARD D, COX D, et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems[J]. Nature Biotechnology, 2013, 31(3): 233-239.
9	LIU X, GALLAY C, KJOS M, et al. High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae [J]. Molecular Systems Biology, 2017, 13(5): 931.
10	WANG T M, GUAN C G, GUO J H, et al. Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance[J]. Nature Communications, 2018, 9: 2475.
11	DONG C, FONTANA J, PATEL A, et al. Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria[J]. Nature Communications, 2018, 9: 2489.
12	ANZALONE A V, KOBLAN L W, LIU D R. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors[J]. Nature Biotechnology, 2020, 38(7): 824-844.
13	CHO S I, LEE S H, MOK YG, et al. Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases[J]. Cell, 2022, 185(10): 1764-1776.e12.
14	DREISSIG S, SCHIML S, SCHINDELE P, et al. Live-cell CRISPR imaging in plants reveals dynamic telomere movements[J]. The Plant Journal, 2017, 91(4): 565-573.
15	MAASS P G, BARUTCU A R, SHECHNER D M, et al. Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging (SNP-CLING)[J]. Nature Structural & Molecular Biology, 2018, 25(2): 176-184.
16	LIU X S, WU H, KRZISCH M, et al. Rescue of fragile X syndrome neurons by DNA methylation editing of the FMR1 gene[J]. Cell, 2018, 172(5): 979-992.e6.
17	KANG J G, PARK J S, KO J H, et al. Regulation of gene expression by altered promoter methylation using a CRISPR/Cas9-mediated epigenetic editing system[J]. Scientific Reports, 2019, 9: 11960.
18	SHMAKOV S, SMARGON A, SCOTT D, et al. Diversity and evolution of class 2 CRISPR-Cas systems[J]. Nature Reviews Microbiology, 2017, 15(3): 169-182.
19	BARRANGOU R, GERSBACH C A. Expanding the CRISPR toolbox: targeting RNA with Cas13b[J]. Molecular Cell, 2017, 65(4): 582-584.
20	PAUSCH P, AL-SHAYEB B, BISOM-RAPP E, et al. CRISPR-CasΦ from huge phages is a hypercompact genome editor[J]. Science, 2020, 369(6501): 333-337.
21	STRECKER J, LADHA A, GARDNER Z, et al. RNA-guided DNA insertion with CRISPR-associated transposases[J]. Science, 2019, 365(6448): 48-53.
22	CASINI A, OLIVIERI M, PETRIS G, et al. A highly specific SpCas9 variant is identified by in vivo screening in yeast[J]. Nature Biotechnology, 2018, 36(3): 265-271.
23	KLEINSTIVER B P, SOUSA A A, WALTON R T, et al. Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing[J]. Nature Biotechnology, 2019, 37(3): 276-282.
24	GAUDELLI N M, KOMOR A C, REES H A, et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage[J]. Nature, 2017, 551(7681): 464-471.
25	HUANG T P, NEWBY G A, LIU D R. Precision genome editing using cytosine and adenine base editors in mammalian cells[J]. Nature Protocols, 2021, 16(2): 1089-1128.
26	LIU R M, LIANG L Y, FREED E F, et al. Directed evolution of CRISPR/cas systems for precise gene editing[J]. Trends in Biotechnology, 2021, 39(3): 262-273.
27	HIRANO S, ABUDAYYEH O O, GOOTENBERG J S, et al. Structural basis for the promiscuous PAM recognition by Corynebacterium diphtheriae Cas9[J]. Nature Communications, 2019, 10: 1968.
28	SWARTS D C, JINEK M. Cas9 versus Cas12a/Cpf1: structure-function comparisons and implications for genome editing[J]. Wiley Interdisciplinary Reviews: RNA, 2018, 9(5): e1481.
29	ANDERS C, NIEWOEHNER O, DUERST A, et al. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease[J]. Nature, 2014, 513(7519): 569-573.
30	SWARTS D C, VAN DER OOST J, JINEK M. Structural basis for guide RNA processing and seed-dependent DNA targeting by CRISPR-Cas12a[J]. Molecular Cell, 2017, 66(2): 221-233.e4.
31	KLEINSTIVER B P, PATTANAYAK V, PREW M S, et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects[J]. Nature, 2016, 529(7587): 490-495.
32	SLAYMAKER I M, GAO L Y, ZETSCHE B, et al. Rationally engineered Cas9 nucleases with improved specificity[J]. Science, 2016, 351(6268): 84-88.
33	CHEN J S, DAGDAS Y S, KLEINSTIVER B P, et al. Enhanced proofreading governs CRISPR-Cas9 targeting accuracy[J]. Nature, 2017, 550(7676): 407-410.
34	BRAVO J P K, LIU M S, HIBSHMAN G N, et al. Structural basis for mismatch surveillance by CRISPR-Cas9[J]. Nature, 2022, 603(7900): 343-347.
35	ZUO Z C, BABU K, GANGULY C, et al. Rational engineering of CRISPR-Cas9 nuclease to attenuate position-dependent off-target effects[J]. The CRISPR Journal, 2022, 5(2): 329-340.
36	TAN Y Y, CHU A H Y, BAO S Y, et al. Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity[J]. Proceedings of the National Academy of Sciences of the United States of America, 2019, 116(42): 20969-20976.
37	LEE J K, JEONG E, LEE J, et al. Directed evolution of CRISPR-Cas9 to increase its specificity[J]. Nature Communications, 2018, 9: 3048.
38	SINGH D, WANG Y B, MALLON J, et al. Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis[J]. Nature Structural & Molecular Biology, 2018, 25(4): 347-354.
39	VAKULSKAS C A, DEVER D P, RETTIG G R, et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells[J]. Nature Medicine, 2018, 24(8): 1216-1224.
40	LIU R M, LIANG L L, FREED E, et al. Synthetic chimeric nucleases function for efficient genome editing[J]. Nature Communications, 2019, 10: 5524.
41	HU J H, MILLER S M, GEURTS M H, et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity[J]. Nature, 2018, 556(7699): 57-63.
42	GUO M H, REN K, ZHU Y W, et al. Structural insights into a high fidelity variant of SpCas9[J]. Cell Research, 2019, 29(3): 183-192.
43	CHOI G C G, ZHOU P, YUEN C T L, et al. Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9[J]. Nature Methods, 2019, 16(8): 722-730.
44	GAO L Y, COX D B T, YAN W X, et al. Engineered Cpf1 variants with altered PAM specificities[J]. Nature Biotechnology, 2017, 35(8): 789-792.
45	TSAI S Q, WYVEKENS N, KHAYTER C, et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing[J]. Nature Biotechnology, 2014, 32(6): 569-576.
46	CHATTERJEE P, JAKIMO N, JACOBSON J M. Minimal PAM specificity of a highly similar SpCas9 ortholog[J]. Science Advances, 2018, 4(10): eaau0766.
47	TÓTH E, CZENE B C, KULCSÁR P I, et al. 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[J]. Nucleic Acids Research, 2018, 46(19): 10272-10285.
48	CONG L, RAN F A, COX D, et al. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013, 339(6121): 819-823.
49	KLEINSTIVER B P, PREW M S, TSAI S Q, et al. Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition[J]. Nature Biotechnology, 2015, 33(12): 1293-1298.
50	MA D C, XU Z M, ZHANG Z Y, et al. Engineer chimeric Cas9 to expand PAM recognition based on evolutionary information[J]. Nature Communications, 2019, 10: 560.
51	ZETSCHE B, GOOTENBERG J S, ABUDAYYEH O O, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system[J]. Cell, 2015, 163(3): 759-771.
52	VILLIGER L, GRISCH-CHAN H M, LINDSAY H, et al. Treatment of a metabolic liver disease by in vivo genome base editing in adult mice[J]. Nature Medicine, 2018, 24(10): 1519-1525.
53	KLEINSTIVER B P, PREW M S, TSAI S Q, et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities[J]. Nature, 2015, 523(7561): 481-485.
54	HIRANO S, NISHIMASU H, ISHITANI R, et al. Structural basis for the altered PAM specificities of engineered CRISPR-Cas9[J]. Molecular Cell, 2016, 61(6): 886-894.
55	HU X X, MENG X B, LIU Q, et al. Increasing the efficiency of CRISPR-Cas9-VQR precise genome editing in rice[J]. Plant Biotechnology Journal, 2018, 16(1): 292-297.
56	NISHIMAS U H, RAN F A, HSU P D, et al. Crystal structure of Casg in complex with guide RNA and target DNA[J]. Cell, 2014, 156(5): 935-949.
57	NISHIMASU H, SHI X, ISHIGURO S, et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space[J]. Science, 2018, 361(6408): 1259-1262.
58	WALTON R T, CHRISTIE K A, WHITTAKER M N, et al. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants[J]. Science, 2020, 368(6488): 290-296.
59	HSU P D, LANDER E S, ZHANG F. Development and applications of CRISPR-Cas9 for genome engineering[J]. Cell, 2014, 157(6): 1262-1278.
60	HIRANO H, GOOTENBERG J S, HORII T, et al. Structure and engineering of Francisella novicida Cas9[J]. Cell, 2016, 164(5): 950-961.
61	YAMAMOTO A, ISHIDA T, YOSHIMURA M, et al. Developing heritable mutations in Arabidopsis thaliana using a modified CRISPR/Cas9 toolkit comprising PAM-altered Cas9 variants and gRNAs[J]. Plant and Cell Physiology, 2019, 60(10): 2255-2262.
62	MILLER S M, WANG T N, RANDOLPH P B, et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs[J]. Nature Biotechnology, 2020, 38(4): 471-481.
63	GUILINGER J P, THOMPSON D B, LIU D R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification[J]. Nature Biotechnology, 2014, 32(6): 577-582.
64	AOUIDA M, EID A, ALI Z, et al. Efficient fdCas9 synthetic endonuclease with improved specificity for precise genome engineering[J]. PLoS One, 2015, 10(7): e0133373.
65	WYVEKENS N, TOPKAR V V, KHAYTER C, et al. Dimeric CRISPR RNA-guided FokI-dCas9 nucleases directed by truncated gRNAs for highly specific genome editing[J]. Human Gene Therapy, 2015, 26(7): 425-431.
66	TERAO M, TAMANO M, HARA S, et al. Utilization of the CRISPR/Cas9 system for the efficient production of mutant mice using crRNA/tracrRNA with Cas9 nickase and FokI-dCas9[J]. Experimental Animals, 2016, 65(3): 275-283.
67	MA D C, PENG S G, HUANG W R, et al. Rational design of mini-Cas9 for transcriptional activation[J]. ACS Synthetic Biology, 2018, 7(4): 978-985.
68	HAVLICEK S, SHEN Y, ALPAGU Y, et al. Re-engineered RNA-guided FokI-nucleases for improved genome editing in human cells[J]. Molecular Therapy, 2017, 25(2): 342-355.
69	KONERMANN S, BRIGHAM M D, TREVINO A E, et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex[J]. Nature, 2015, 517(7536): 583-588.
70	PEREZ-PINERA P, KOCAK D D, VOCKLEY C M, et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors[J]. Nature Methods, 2013, 10(10): 973-976.
71	YEO N C, CHAVEZ A, LANCE-BYRNE A, et al. An enhanced CRISPR repressor for targeted mammalian gene regulation[J]. Nature Methods, 2018, 15(8): 611-616.
72	LIAN J Z, HAMEDIRAD M, HU S M, et al. Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system[J]. Nature Communications, 2017, 8: 1688.
73	BEHLER J, VIJAY D, HESS W R. CRISPR-based technologies for metabolic engineering in cyanobacteria[J]. Trends in Biotechnology, 2018, 36(10): 996-1010.
74	TONG Y J, CHARUSANTI P, ZHANG L X, et al. CRISPR-Cas9 based engineering of actinomycetal genomes[J]. ACS Synthetic Biology, 2015, 4(9): 1020-1029.
75	SCHMIDT R, STEINHART Z, LAYEGHI M, et al. CRISPR activation and interference screens decode stimulation responses in primary human T cells[J]. Science, 2022, 375(6580): eabj4008.
76	KOMOR A C, KIM Y B, PACKER M S, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage[J]. Nature, 2016, 533(7603): 420-424.
77	KOMOR A C, ZHAO K T, PACKER M S, 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[J]. Science Advances, 2017, 3(8): eaao4774.
78	ZHAO D D, LI J, LI S W, et al. Glycosylase base editors enable C-to-A and C-to-G base changes[J]. Nature Biotechnology, 2021, 39(1): 35-40.
79	KOBLAN L W, ARBAB M, SHEN M W, et al. Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library analysis, and machine learning[J]. Nature Biotechnology, 2021, 39(11): 1414-1425.
80	ANZALONE A V, RANDOLPH P B, DAVIS J R, et al. Search-and-replace genome editing without double-strand breaks or donor DNA[J]. Nature, 2019, 576(7785): 149-157.
81	NELSON J W, RANDOLPH P B, SHEN S P, et al. Engineered pegRNAs improve prime editing efficiency[J]. Nature Biotechnology, 2022, 40(3): 402-410.
82	CHEN P J, HUSSMANN J A, YAN J, et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes[J]. Cell, 2021, 184(22): 5635-5652.e29.
83	LIN Q P, JIN S, ZONG Y, et al. High-efficiency prime editing with optimized, paired pegRNAs in plants[J]. Nature Biotechnology, 2021, 39(8): 923-927.
84	CHOI J, CHEN W, SUITER C C, et al. Precise genomic deletions using paired prime editing[J]. Nature Biotechnology, 2022, 40(2): 218-226.
85	ANZALONE A V, GAO X D, PODRACKY C J, et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing[J]. Nature Biotechnology, 2022, 40(5): 731-740.
86	KOBLAN L W, DOMAN J L, WILSON C, et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction[J]. Nature Biotechnology, 2018, 36(9): 843-846.
87	ARBAB M, SHEN M W, MOK B, et al. Determinants of base editing outcomes from target library analysis and machine learning[J]. Cell, 2020, 182(2): 463-480.e30.
88	WIJSMAN M, ŚWIAT M A, MARQUES W L, et al. A toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficient Saccharomyces cerevisiae strains[J]. FEMS Yeast Research, 2018, 19(1): foy107.
89	YUAN Q C, GAO X. Multiplex base- and prime-editing with drive-and-process CRISPR arrays[J]. Nature Communications, 2022, 13: 2771.
90	RAUCH B J, SILVIS M R, HULTQUIST J F, et al. Inhibition of CRISPR-Cas9 with bacteriophage proteins[J]. Cell, 2017, 168(1/2): 150-158.e10.
91	HE F, BHOOBALAN-CHITTY Y, VAN L B, et al. Anti-CRISPR proteins encoded by archaeal lytic viruses inhibit subtype I-D immunity[J]. Nature Microbiology, 2018, 3(4): 461-469.
92	STANLEY S Y, MAXWELL K L. Phage-encoded anti-CRISPR defenses[J]. Annual Review of Genetics, 2018, 52: 445-464.
93	OSUNA B A, KARAMBELKAR S, MAHENDRA C, et al. Listeria phages induce Cas9 degradation to protect lysogenic genomes[J]. Cell Host & Microbe, 2020, 28(1): 31-40.e9.
94	LIU L, YIN M L, WANG M, et al. Phage AcrIIA2 DNA mimicry: Structural basis of the CRISPR and anti-CRISPR arms race[J]. Molecular Cell, 2019, 73(3): 611-620.e3.
95	YANG H, PATEL D J. Inhibition mechanism of an anti-CRISPR suppressor AcrIIA4 targeting SpyCas9[J]. Molecular Cell, 2017, 67(1): 117-127.e5.
96	WATTERS K E, SHIVRAM H, FELLMANN C, et al. Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes[J]. Proceedings of the National Academy of Sciences of the United States of America, 2020, 117(12): 6531-6539.
97	WATTERS K E, FELLMANN C, BAI H B, et al. Systematic discovery of natural CRISPR-Cas12a inhibitors[J]. Science, 2018, 362(6411): 236-239.
98	MARINO N D, ZHANG J Y, BORGES A L, et al. Discovery of widespread type I and type V CRISPR-Cas inhibitors[J]. Science, 2018, 362(6411): 240-242.
99	KNOTT G J, THORNTON B W, LOBBA M J, et al. Broad-spectrum enzymatic inhibition of CRISPR-Cas12a[J]. Nature Structural & Molecular Biology, 2019, 26(4): 315-321.
100	PENG R C, LI Z T, XU Y, et al. Structural insight into multistage inhibition of CRISPR-Cas12a by AcrVA4[J]. Proceedings of the National Academy of Sciences of the United States of America, 2019, 116(38): 18928-18936.
101	DONG L Y, GUAN X Y, LI N N, et al. An anti-CRISPR protein disables type V Cas12a by acetylation[J]. Nature Structural & Molecular Biology, 2019, 26(4): 308-314.
102	SHIN J, JIANG F G, LIU J J, et al. Disabling Cas9 by an anti-CRISPR DNA mimic[J]. Science Advances, 2017, 3(7): e1701620.
103	LIANG M M, SUI T T, LIU Z Q, et al. AcrIIA5 suppresses base editors and reduces their off-target effects[J]. Cells, 2020, 9(8): 1786.
104	LI C, PSATHA N, GIL S, et al. HDAd5/35⁺⁺ adenovirus vector expressing anti-CRISPR peptides decreases CRISPR/Cas9 toxicity in human hematopoietic stem cells[J]. Molecular Therapy-Methods & Clinical Development, 2018, 9: 390-401.
105	LI J, XU Z L, CHUPALOV A, et al. Anti-CRISPR-based biosensors in the yeast S. cerevisiae [J]. Journal of Biological Engineering, 2018, 12: 11.
106	BASGALL E M, GOETTING S C, GOECKEL M E, et al. Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in Saccharomyces cerevisiae [J]. Microbiology, 2018, 164(4): 464-474.
107	BUBECK F, HOFFMANN M D, HARTEVELD Z, et al. Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9[J]. Nature Methods, 2018, 15(11): 924-927.
108	JOHNSTON R K, SEAMON K J, SAADA E A, et al. Use of anti-CRISPR protein AcrIIA4 as a capture ligand for CRISPR/Cas9 detection[J]. Biosensors & Bioelectronics, 2019, 141: 111361.
109	JIANG F G, DOUDNA J A. CRISPR-cas9 structures and mechanisms[J]. Annual Review of Biophysics, 2017, 46: 505-529.
110	NIHONGAKI Y, OTABE T, UEDA Y, et al. A split CRISPR-Cpf1 platform for inducible genome editing and gene activation[J]. Nature Chemical Biology, 2019, 15(9): 882-888.
111	YU Y H, WU X, GUAN N Z, et al. Engineering a far-red light-activated split-Cas9 system for remote-controlled genome editing of internal organs and tumors[J]. Science Advances, 2020, 6(28): eabb1777.
112	SUNG K, PARK J, KIM Y, et al. Target specificity of Cas9 nuclease via DNA rearrangement regulated by the REC2 domain[J]. Journal of the American Chemical Society, 2018, 140(25): 7778-7781.
113	LU Y, XUE J X, DENG T, et al. Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer[J]. Nature Medicine, 2020, 26(5): 732-740.
114	MA S F, WANG X L, HU Y F, et al. Enhancing site-specific DNA integration by a Cas9 nuclease fused with a DNA donor-binding domain[J]. Nucleic Acids Research, 2020, 48(18): 10590-10601.
115	YIN J H, LU R S, XIN C C, et al. Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing[J]. Nature Communications, 2022, 13: 1204.
116	ZUO E W, SUN Y D, WEI W, et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos[J]. Science, 2019, 364(6437): 289-292.
117	YIN H, SONG C Q, SURESH S, et al. Partial DNA-guided Cas9 enables genome editing with reduced off-target activity[J]. Nature Chemical Biology, 2018, 14(3): 311-316.
118	KOCAK D D, JOSEPHS E A, BHANDARKAR V, et al. Increasing the specificity of CRISPR systems with engineered RNA secondary structures[J]. Nature Biotechnology, 2019, 37(6): 657-666.

名称	突变	改造的结构域	PAM	精度最高提升倍数	参考文献
SpCas9-HF1	N497A/R661A/Q695A/Q926A	REC3, RuvC3	NGG	19	[31]
eSpCas9(1.1)	K848A/K1003A/R1060A	HNH, RuvC3 (nt-groove)	NGG	19	[32-33]
HypaCas9	N692A/M694A/Q695A/H698A	REC3	NGG	19	[33]
SuperFi-Cas9	Y1010D/Y1013D/Y1016D/V1018D/R1019D/Q1027D/K1031D	RuvC	NGG	4.06^②	[34]
hscCas9-v1.2	N14A/R765A/R447A/S845D	REC1, RuvC, HNH	NGG	1.67	[35]
SaCas9-HF	R245A/N413A/N419A/R654A	REC, RuvC3	NNGRRT	2	[36]
enAsCas12a-HF1	N282A^①	REC1	TTYN, VTTV, TRTV	1.89	[23]

名称	突变	改造的结构域	PAM	精度最高提升倍数	参考文献
SpCas9-HF1	N497A/R661A/Q695A/Q926A	REC3, RuvC3	NGG	19	[31]
eSpCas9(1.1)	K848A/K1003A/R1060A	HNH, RuvC3 (nt-groove)	NGG	19	[32-33]
HypaCas9	N692A/M694A/Q695A/H698A	REC3	NGG	19	[33]
SuperFi-Cas9	Y1010D/Y1013D/Y1016D/V1018D/R1019D/Q1027D/K1031D	RuvC	NGG	4.06^②	[34]
hscCas9-v1.2	N14A/R765A/R447A/S845D	REC1, RuvC, HNH	NGG	1.67	[35]
SaCas9-HF	R245A/N413A/N419A/R654A	REC, RuvC3	NNGRRT	2	[36]
enAsCas12a-HF1	N282A^①	REC1	TTYN, VTTV, TRTV	1.89	[23]

名称	突变	改造的结构域	PAM	精度最高提升倍数	参考文献
evoCas9	M495V/Y515N/K526E/H698Q	REC3	NGG	9	[22]
HiFi Cas9	R691A	REC3	NGG	2.46	[39]
xCas9-3.7	A262T/R324L/S409I/E480K/E543D/M694I/E1219V^①	REC1, REC2, REC3, PI	NG, NNG, GAA, GAT and CAA	0.29	[41-42]
Sniper-Cas9	F539S/M763I/K890N	REC3, RuvC2, HNH	NGG	8.47	[37]
Opti-SpCas9	R661A/K1003H	REC3, RuvC3	NGG	5	[43]
AsCas12a-K949A	K949A	BH	TTTV	0.23	[44]
M44	TX_Cas12a的REC1结构域替换	REC1	TTTN	24	[40]

名称	突变	改造的结构域	PAM	精度最高提升倍数	参考文献
evoCas9	M495V/Y515N/K526E/H698Q	REC3	NGG	9	[22]
HiFi Cas9	R691A	REC3	NGG	2.46	[39]
xCas9-3.7	A262T/R324L/S409I/E480K/E543D/M694I/E1219V^①	REC1, REC2, REC3, PI	NG, NNG, GAA, GAT and CAA	0.29	[41-42]
Sniper-Cas9	F539S/M763I/K890N	REC3, RuvC2, HNH	NGG	8.47	[37]
Opti-SpCas9	R661A/K1003H	REC3, RuvC3	NGG	5	[43]
AsCas12a-K949A	K949A	BH	TTTV	0.23	[44]
M44	TX_Cas12a的REC1结构域替换	REC1	TTTN	24	[40]

名称	突变	改造的结构域	PAM范围变化	参考文献
SpCas9^①			NGG	[48]
xCas9-3.7	A262T/R324L/S409I/E480K/E543D/M694I/E1219V	REC1, REC2, REC3, PI	NG, NNG, GAA, GAT and CAA	[41]
SaCas9^①			NNGRRT^②	[8]
SaCas9 KKH	E782K/N968K/R1015H	PI	NNNRRT	[49]
v42-wt	SaCas9 上PI结构域的13个保守氨基酸序列被替换	PI	NNVRRN^②	[50]
AsCas12a/LbCas12a^①			TTTV	[51]
AsCas12a	S542R/K607R	WED, PI	TYCV^②	[44, 52]
AsCas12a	S542R/K548V/N552R	WED, PI	TATV	[44]
LbCas12a	G532R/K595R	WED, PI	TYCV	[44, 52]
LbCas12a	G532R/K538V/Y542R	WED, PI	TATV	[44]

靶向DNA的Ⅱ类CRISPR/Cas系统的蛋白工程化改造

Protein engineering of DNA targeting type Ⅱ CRISPR/Cas systems

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名称	突变	改造的结构域	PAM范围变化	参考文献
SpCas9^①			NGG	[48]
SpCas9 VQR	D1135V/R1335Q/T1337R	WED, PI	NGAN NGCG	[53-55]
SpCas9 EQR	D1135E/R1335Q/T1337R	WED, PI	NGAG	[53-54]
SpCas9 VRER	D1135V/G1218R/R1335E/T1337R	WED, PI	NGCG	[53-54]
Sp-St3Cas9	SpCas9 与St3Cas9的PI结构域替换	PI	NGGNG	[56]
SpCas9-NG	R1335V/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R	WED, PI	NG	[57]
SpG	D1135L/S1136W/G1218K/E1219Q/R1335Q/T1337R	WED, PI	NGN	[58]
SpRY	SpG(A61R/L1111R/A1322R/ /N1317R/ R1333P)	BH, WED, PI	NRN	[58]
FnCas9^①			NGG	[59]
RHA FnCas9	E1369R/E1449H/R1556A	WED, PI	YG^②	[60]

名称	CRISPR/Cas	Base editor	PE
Class/Type	Type Ⅱ	Type Ⅱ	Type Ⅱ
靶向序列长度/bp	19～23	15～20	8～15
DNA编辑方式	有DSB的HDR	无DSB的碱基替换	无DSB的HDR
On-target	高	较高	低
Off-target	较低	低	低
多位点编辑	可达9位点^[88]	可达31位点^[89]	可达3位点^[89]
应用范围	人体、动物、植物、微生物	人体、动物、植物、微生物	人体、动物、植物、微生物

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