基于CRISPR/Cas系统的多重基因编辑与调控技术

doi:10.13345/j.cjb.220989

科微学术

生物工程学报

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基于CRISPR/Cas系统的多重基因编辑与调控技术
DOI:
                        10.13345/j.cjb.220989
                    
CSTR:
                        32114.14.j.cjb.220989
                    
作者:
                        樊祥瑞樊祥瑞
大连理工大学生物工程学院, 辽宁 大连 116000
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王俊燕王俊燕
大连理工大学生物工程学院, 辽宁 大连 116000
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梁丽亚梁丽亚
大连理工大学生物工程学院, 辽宁 大连 116000
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刘嵘明刘嵘明
大连理工大学生物工程学院, 辽宁 大连 116000
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基金项目:国家自然科学基金(22278058， 22208044)；大连理工大学基本科研业务费(DUT22RC(3)012， DUT22RC(3)013)

Multiplex gene editing and regulation techniques based on CRISPR/Cas system

Author:

FAN Xiangrui
FAN Xiangrui
School of Bioengineering, Dalian University of Technology, Dalian 116000, Liaoning, China
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WANG Junyan
WANG Junyan
School of Bioengineering, Dalian University of Technology, Dalian 116000, Liaoning, China
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LIANG Liya
LIANG Liya
School of Bioengineering, Dalian University of Technology, Dalian 116000, Liaoning, China
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LIU Rongming
LIU Rongming
School of Bioengineering, Dalian University of Technology, Dalian 116000, Liaoning, China
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参考文献 [76]

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摘要:

规律成簇的间隔短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)及其相关Cas蛋白所构建的CRISPR/Cas系统是古细菌或细菌中特有的一种获得性免疫系统。研究人员将其开发成基因编辑工具之后，凭借其高效、精准和通用性强等优点迅速成为合成生物学领域的热门研究方向，在生命科学、生物工程技术、食品科学及农作物育种等多个领域引发了革命性的影响。目前基于CRISPR/Cas系统单基因编辑与调控技术日益完善，但在多重基因编辑和调控方面仍存在挑战。本文聚焦基于CRISPR/Cas系统的多重基因编辑与调控技术开发及应用，针对单个细胞内实现多位点基因编辑或调控和细胞群体内实现多位点基因编辑或调控技术，依据作用原理对其进行了系统总结和阐述，包括基于CRISPR/Cas系统的双链断裂、单链断裂以及多重基因调控技术等。这些工作丰富了多重基因编辑与调控的工具，为CRISPR/Cas系统在多领域的应用作出了贡献。

关键词:规律成簇的间隔短回文重复序列及其相关Cas蛋白;可追踪基因编辑技术;碱基编辑器;先导编辑器;基因激活;基因抑制

Abstract:

The CRISPR/Cas systems comprising the clustered regularly interspaced short palindromic repeats (CRISPR) and its associated Cas protein is an acquired immune system unique to archaea or bacteria. Since its development as a gene editing tool, it has rapidly become a popular research direction in the field of synthetic biology due to its advantages of high efficiency, precision, and versatility. This technique has since revolutionized the research of many fields including life sciences, bioengineering technology, food science, and crop breeding. Currently, the single gene editing and regulation techniques based on CRISPR/Cas systems have been increasingly improved, but challenges still exist in the multiplex gene editing and regulation. This review focuses on the development and application of multiplex gene editing and regulation techniques based on the CRISPR/Cas systems, and summarizes the techniques for multiplex gene editing or regulation within a single cell or within a cell population. This includes the multiplex gene editing techniques developed based on the CRISPR/Cas systems with double-strand breaks; or with single-strand breaks; or with multiple gene regulation techniques, etc. These works have enriched the tools for the multiplex gene editing and regulation and contributed to the application of CRISPR/Cas systems in the multiple fields.

Key words:CRISPR/Cas;CRISPR-enabled trackable genome engineering (CREATE) technology;base editors;prime editors;CRISPR activation;CRISPR interference

参考文献

[1] GASIUNAS G, BARRANGOU R, HORVATH P, SIKSNYS V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(39): E2579-E2586.

[2] CARROLL D. Genome engineering with zinc-finger nucleases[J]. Genetics, 2011, 188(4): 773-782.

[3] NEMUDRYI AA, VALETDINOVA KR, MEDVEDEV SP, ZAKIAN SM. TALEN and CRISPR/Cas genome editing systems: tools of discovery[J]. Acta Naturae, 2014, 6(3): 19-40.

[4] GAJ T, GERSBACH CA, BARBAS CF, 3RD. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering[J]. Trends in Biotechnology, 2013, 31(7): 397-405.

[5] WANG HX, LI M, LEE CM, CHAKRABORTY S, KIM HW, BAO G, LEONG KW. CRISPR/Cas9-based genome editing for disease modeling and therapy: challenges and opportunities for nonviral delivery[J]. Chemical Reviews, 2017, 117(15): 9874-9906.

[6] JIANG W, BIKARD D, COX D, ZHANG F, MARRAFFINI LA. RNA-guided editing of bacterial genomes using CRISPR-Cas systems[J]. Nature Biotechnology, 2013, 31(3): 233-239.

[7] QI L S, LARSON MH, GILBERT LA, DOUDNA JA, WEISSMAN JS, ARKIN AP, LIM WA. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression[J]. Cell, 2013, 152(5): 1173-1183.

[8] GILBERT LA, LARSON MH, MORSUT L, LIU Z, BRAR GA, TORRES SE, STERN-GINOSSAR N, BRANDMAN O, WHITEHEAD EH, DOUDNA JA, LIM WA, WEISSMAN JS, QI LS. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes[J]. Cell, 2013, 154(2): 442-451.

[9] DREISSIG S, SCHIML S, SCHINDELE P, WEISS O, RUTTEN T, SCHUBERT V, GLADILIN E, METTE MF, PUCHTA H, HOUBEN A. Live-cell CRISPR imaging in plants reveals dynamic telomere movements[J]. Plant Journal, 2017, 91(4): 565-573.

[10] KANG JG, PARK JS, KO JH, KIM YS. Regulation of gene expression by altered promoter methylation using a CRISPR/Cas9-mediated epigenetic editing system[J]. Scientific Reports, 2019, 9(1): 1-12.

[11] MALI P, YANG LH, ESVELT KM, AACH J, GUELL M, DICARLO JE, NORVILLE JE, CHURCH GM. RNA-guided human genome engineering via Cas9[J]. Science, 2013, 339(6121): 823-826.

[12] SHMAKOV S, SMARGON A, SCOTT D, COX D, PYZOCHA N, YAN W, ABUDAYYEH OO, GOOTENBERG JS, MAKAROVA KS, WOLF YI, SEVERINOV K, ZHANG F, KOONIN EV. Diversity and evolution of class 2 CRISPR-Cas systems[J]. Nature Reviews Microbiology, 2017, 15(3): 169-182.

[13] BARRANGOU R, GERSBACH CA. Expanding the CRISPR toolbox: targeting RNA with Cas13b[J]. Molecular Cell, 2017, 65(4): 582-584.

[14] CONG L, RAN FA, COX D, LIN SL, BARRETTO R, HABIB N, HSU PD, WU XB, JIANG WY, MARRAFFINI LA, ZHANG F. Multiplex genome engineering using CRISPR/Cas systems[J]. Science, 2013, 339(6121): 819-823.

[15] KOMOR AC, KIM YB, PACKER MS, ZURIS JA, LIU DR. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage[J]. Nature, 2016, 533(7603): 420-424.

[16] REES HA, LIU DR. Base editing: precision chemistry on the genome and transcriptome of living cells[J]. Nature Reviews Genetics, 2018, 19(12): 770-788.

[17] GAUDELLI NM, KOMOR AC, REES HA, PACKER MS, BADRAN AH, BRYSON DI, LIU DR. Programmable base editing of A.T to G.C in genomic DNA without DNA cleavage[J]. Nature, 2017, 551(7681): 464-471.

[18] LIN Q, ZONG Y, XUE C, WANG S, JIN S, ZHU Z, WANG Y, ANZALONE AV, RAGURAM A, DOMAN JL, LIU DR, GAO C. Prime genome editing in rice and wheat[J]. Nature Biotechnology, 2020, 38(5): 582-585.

[19] ANZALONE AV, KOBLAN LW, LIU DR. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors[J]. Nature Biotechnology, 2020, 38(7): 824-844.

[20] SANDER JD, JOUNG JK. CRISPR-Cas systems for editing, regulating and targeting genomes[J]. Nature Biotechnology, 2014, 32(4): 347-355.

[21] GILBERT LA, HORLBECK MA, ADAMSON B, VILLALTA JE, CHEN Y, WHITEHEAD EH, GUIMARAES C, PANNING B, PLOEGH HL, BASSIK MC, QI LS, KAMPMANN M, WEISSMAN JS. Genome-scale CRISPR-mediated control of gene repression and activation[J]. Cell, 2014, 159(3): 647-661.

[22] TSAI SQ, WYVEKENS N, KHAYTER C, FODEN JA, THAPAR V, REYON D, GOODWIN MJ, ARYEE MJ, JOUNG JK. Dimeric CRISPR RNA-guided Fokl nucleases for highly specific genome editing[J]. Nature Biotechnology, 2014, 32(6): 569-576.

[23] BAO Z, XIAO H, LIANG J, ZHANG L, XIONG X, SUN N, SI T, ZHAO H. Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae[J]. ACS Synthetic Biology, 2015, 4(5): 585-594.

[24] XIE KB, MINKENBERG B, YANG YN. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system[J]. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(11): 3570-3575.

[25] JIANG Y, CHEN B, DUAN CL, SUN BB, YANG JJ, YANG S. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system[J]. Applied and Environmental Microbiology, 2015, 81(7): 2506-2514.

[26] ZHANG YP, WANG J, WANG ZB, ZHANG YM, SHI SB, NIELSEN J, LIU ZH. A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae[J]. Nature Communications, 2019, 10(1): 1053.

[27] HORWITZ AA, WALTER JM, SCHUBERT MG, KUNG SH, HAWKINS K, PLATT DM, HERNDAY AD, MAHATDEJKUL-MEADOWS T, SZETO W, CHANDRAN SS, NEWMAN JD. Efficient multiplexed integration of synergistic alleles and metabolic pathways in yeasts via CRISPR-Cas[J]. Cell Systems, 2015, 1(1): 88-96.

[28] WANG LY, DENG AH, ZHANG Y, LIU SW, LIANG Y, BAI H, CUI D, QIU QD, SHANG XL, YANG Z, HE XP, WEN TY. Efficient CRISPR-Cas9 mediated multiplex genome editing in yeasts[J]. Biotechnology for Biofuels, 2018, 11: 1-16.

[29] GUO SY, GAO G, ZHANG CZ, PENG G. Multiplexed genome editing for efficient phenotypic screening in Zebrafish[J]. Veterinary Sciences, 2022, 9(2): 92.

[30] WANG Y, LIU Y, LIU J, GUO YM, FAN LW, NI XM, ZHENG XM, WANG M, ZHENG P, SUN JB, MA YH. MACBETH: Multiplex automated Corynebacterium glutamicum base editing method[J]. Metabolic Engineering, 2018, 47: 200-210.

[31] WANG MG, MAO YF, LU YM, WANG ZD, TAO XP, ZHU JK. Multiplex gene editing in rice with simplified CRISPR-Cpf1 and CRISPR-Cas9 systems[J]. Journal of Integrative Plant Biology, 2018, 60(8): 626-631.

[32] CHEN FB, LIAN M, MA BX, GOU SX, LUO X, YANG KM, SHI H, XIE JK, GE WK, OUYANG Z, LAI CD, LI N, ZHANG QJ, JIN Q, LIANG YH, CHEN T, WANG JW, ZHAO XZ, LI L, YU MY, et al. Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors[J]. Communications Biology, 2022, 5(1): 1163.

[33] FENG X, ZHAO DD, ZHANG XL, DING X, BI CH. CRISPR/Cas9 assisted multiplex genome editing technique in Escherichia coli[J]. Biotechnology Journal, 2018, 13(9): e1700604.

[34] AO X, YAO Y, LI T, YANG TT, DONG X, ZHENG ZT, CHEN GQ, WU Q, GUO YY. A multiplex genome editing method for Escherichia coli based on CRISPR-Cas12a[J]. Frontiers in Microbiology, 2018, 9: 2307.

[35] LI L, WEI KK, ZHENG GS, LIU XC, CHEN SX, JIANG WH, LU YH. CRISPR-Cpf1-assisted multiplex genome editing and transcriptional repression in Streptomyces[J]. Applied and Environmental Microbiology, 2018, 84(18): e00827-18.

[36] FERREIRA R, SKREKAS C, NIELSEN J, DAVID F. Multiplexed CRISPR/Cas9 genome editing and gene regulation using Csy4 in Saccharomyces cerevisiae[J]. Acs Synthetic Biology, 2018, 7(1): 10-15.

[37] MANS R, van ROSSUM HM, WIJSMAN M, BACKX A, KUIJPERS NGA, van den BROEK M, DARAN-LAPUJADE P, PRONK JT, van MARIS AJA, DARAN JMG. CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae[J]. Fems Yeast Research, 2015, 15(2): fov004.

[38] SWIAT MA, DASHKO S, DEN RIDDER M, WIJSMAN M, van der OOST J, DARAN JM, DARAN-LAPUJADE P. FnCpf1: a novel and efficient genome editing tool for Saccharomyces cerevisiae[J]. Nucleic Acids Research, 2017, 45(21): 12585-12598.

[39] KLOMPE SE, VO PLH, HALPIN-HEALY TS, STERNBERG SH. Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration[J]. Nature, 2019, 571(7764): 219-225.

[40] STRECKER J, LADHA A, GARDNER Z, SCHMID-BURGK JL, MAKAROVA KS, KOONIN EV, ZHANG F. RNA-guided DNA insertion with CRISPR-associated transposases[J]. Science, 2019, 365(6448): 48-53.

[41] ZHANG YW, SUN XM, WANG QZ, XU JQ, DONG F, YANG SQ, YANG JW, ZHANG ZX, QIAN Y, CHEN J, ZHANG J, LIU YM, TAO RS, JIANG Y, YANG JJ, YANG S. Multicopy chromosomal integration using CRISPR-associated transposases[J]. Acs Synthetic Biology, 2020, 9(8): 1998-2008.

[42] ZHANG YW, YANG JW, YANG SQ, ZHANG JZ, CHEN J, TAO RS, JIANG Y, YANG JJ, YANG S. Programming cells by multicopy chromosomal integration using CRISPR-associated transposases[J]. Crispr Journal, 2021, 4(3): 350-359.

[43] YANG SQ, ZHANG YW, XU JQ, ZHANG J, ZHANG JZ, YANG JJ, JIANG Y, YANG S. Orthogonal CRISPR-associated transposases for parallel and multiplexed chromosomal integration[J]. Nucleic Acids Research, 2021, 49(17): 10192-10202.

[44] WANG HH, ISAACS FJ, CARR PA, SUN ZZ, XU G, FOREST CR, CHURCH GM. Programming cells by multiplex genome engineering and accelerated evolution[J]. Nature, 2009, 460(7257): 894-U133.

[45] ISAACS FJ, CARR PA, WANG HH, LAJOIE MJ, STERLING B, KRAAL L, TOLONEN AC, GIANOULIS TA, GOODMAN DB, REPPAS NB, EMIG CJ, BANG D, HWANG SJ, JEWETT MC, JACOBSON JM, CHURCH GM. Precise manipulation of chromosomes in vivo enables genome-wide codon replacement[J]. Science, 2011, 333(6040): 348-353.

[46] WARNER JR, REEDER PJ, KARIMPOUR-FARD A, WOODRUFF LBA, GILL RT. Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides[J]. Nature Biotechnology, 2010, 28(8): 856-U138.

[47] GARST AD, BASSALO MC, PINES G, LYNCH SA, HALWEG-EDWARDS AL, LIU R, LIANG L, WANG Z, ZEITOUN R, ALEXANDER WG, GILL RT. Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering[J]. Nature Biotechnology, 2017, 35(1): 48-55.

[48] BASSALO MC, GARST AD, CHOUDHURY A, GRAU WC, OH EJ, SPINDLER E, LIPSCOMB T, GILL RT. Deep scanning lysine metabolism in Escherichia coli[J]. Molecular Systems Biology, 2018, 14(11): e8371.

[49] ZHENG Y, KONG S, LUO S, CHEN C, CUI Z, SUN X, CHEN T, WANG Z. Improving furfural tolerance of Escherichia coli by integrating adaptive laboratory evolution with CRISPR-enabled trackable genome engineering (CREATE)[J]. ACS Sustainable Chemistry & Engineering, 2022, 10(7): 2318-2330.

[50] LIU R, LIANG L, FREED EF, CHOUDHURY A, ECKERT CA, GILL RT. Engineering regulatory networks for complex phenotypes in E. coli[J]. Nature Communications, 2020, 11(1): 4050.

[51] LIU R, LIANG L, CHOUDHURY A, GARST AD, ECKERT CA, OH EJ, WINKLER J, GILL RT. Multiplex navigation of global regulatory networks (MINR) in yeast for improved ethanol tolerance and production[J]. Metabolic Engineering, 2019, 51: 50-58.

[52] LIU R, LIANG L, CHOUDHURY A, BASSALO MC, GARST AD, TARASAVA K, GILL RT. Iterative genome editing of Escherichia coli for 3-hydroxypropionic acid production[J]. Metabolic Engineering, 2018, 47: 303-313.

[53] LIU R, LIANG L, GARST A D, CHOUDHURY A, NOGUE V SI, BECKHAM GT, GILL RT. Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering[J]. Metabolic Engineering, 2018, 47: 10-20.

[54] TONG YJ, WHITFORD CM, ROBERTSEN HL, BLIN K, JORGENSEN TS, KLITGAARD AK, GREN T, JIANG XL, WEBER T, LEE SY. Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST[J]. Proceedings of the National Academy of Sciences of the United States of America, 2019, 116(41): 20366-20375.

[55] BANNO S, NISHIDA K, ARAZOE T, MITSUNOBU H, KONDO A. Deaminase-mediated multiplex genome editing in Escherichia coli[J]. Nature Microbiology, 2018, 3(4): 423-429.

[56] DAVIS JR, WANG X, WITTE IP, HUANG TP, LEVY JM, RAGURAM A, BANSKOTA S, SEIDAH NG, MUSUNURU K, LIU DR. Efficient in vivo base editing via single adeno-associated viruses with size-optimized genomes encoding compact adenine base editors[J]. Nature Biomedical Engineering, 2022, 6(11): 1272-1283.

[57] YUAN QC, GAO X. Multiplex base- and prime-editing with drive-and-process CRISPR arrays[J]. Nature Communications, 2022, 13(1): 2771.

[58] ANZALONE AV, RANDOLPH PB, DAVIS JR, SOUSA AA, KOBLAN LW, LEVY JM, CHEN PJ, WILSON C, NEWBY GA, RAGURAM A, LIU DR. Search-and-replace genome editing without double-strand breaks or donor DNA[J]. Nature, 2019, 576(7785): 149-157.

[59] REIS AC, HALPER SM, VEZEAU GE, CETNAR DP, HOSSAIN A, CLAUER PR, SALIS HM. Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays[J]. Nature Biotechnology, 2019, 37(11): 1294-1301.

[60] MCCARTY NS, SHAW WM, ELLIS T, LEDESMA-AMARO R. Rapid assembly of gRNA arrays via modular cloning in yeast[J]. Acs Synthetic Biology, 2019, 8(4): 906-910.

[61] ZHAO YW, LI L, ZHENG GS, JIANG WH, DENG ZX, WANG ZJ, LU YH. CRISPR/dCas9-mediated multiplex gene repression in Streptomyces[J]. Biotechnology Journal, 2018, 13(9): e1800121.

[62] KACZMARZYK D, CENGIC I, YAO L, HUDSON EP. Diversion of the long-chain acyl-ACP pool in Synechocystis to fatty alcohols through CRISPRi repression of the essential phosphate acyltransferase PlsX[J]. Metabolic Engineering, 2018, 45: 59-66.

[63] ZHANG X, WANG J, CHENG Q, ZHENG X, ZHAO G, WANG J. Multiplex gene regulation by CRISPR-ddCpf1[J]. Cell Discovery, 2017, 3: 17018.

[64] CAMPA CC, WEISBACH NR, SANTINHA AJ, INCARNATO D, PLATT RJ. Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts[J]. Nature Methods, 2019, 16(9): 887-893.

[65] CHENG AW, WANG H, YANG H, SHI L, KATZ Y, THEUNISSEN TW, RANGARAJAN S, SHIVALILA CS, DADON DB, JAENISCH R. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system[J]. Cell Research, 2013, 23(10): 1163-1171.

[66] LIAN J, HAMEDIRAD M, HU S, ZHAO H. Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system[J]. Nature Communications, 2017, 8(1): 1688.

[67] LIAN JZ, SCHULTZ C, CAO MF, HAMEDIRAD M, ZHAO HM. Multi-functional genome-wide CRISPR system for high throughput genotype-phenotype mapping[J]. Nature Communications, 2019, 10(1): 5794.

[68] WANG T, GUAN C, GUO J, LIU B, WU Y, XIE Z, ZHANG C, XING XH. Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance[J]. Nature Communications, 2018, 9(1): 2475.

[69] ROUSSET F, CUI L, SIOUVE E, BECAVIN C, DEPARDIEU F, BIKARD D. Genome-wide CRISPR-dCas9 screens in E. coli identify essential genes and phage host factors[J]. PLos Genetics, 2018, 14(11): e1007749.

[70] SCHMIDT R, STEINHART Z, LAYEGHI M, FREIMER JW, BUENO R, NGUYEN VQ, BLAESCHKE F, YE CJ, MARSON A. CRISPR activation and interference screens decode stimulation responses in primary human T cells[J]. Science, 2022, 375(6580): eabj4008.

[71] SANSON KR, HANNA RE, HEGDE M, DONOVAN KF, STRAND C, SULLENDER ME, VAIMBERG EW, GOODALE A, ROOT DE, PICCIONI F, DOENCH JG. Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities[J]. Nature Communications, 2018, 9(1): 5416.

[72] WANG Y, CHENG HJ, LIU Y, LIU Y, WEN X, ZHANG K, NI XM, GAO N, FAN LW, ZHANG ZH, LIU J, CHEN JZ, WANG LX, GUO YM, ZHENG P, WANG M, SUN JB, MA YH. In-situ generation of large numbers of genetic combinations for metabolic reprogramming via CRISPR-guided base editing[J]. Nature Communications, 2021, 12(1): 678.

[73] LIU R, LIANG L, FREED EF, GILL RT. Directed evolution of CRISPR/Cas systems for precise gene editing[J]. Trends in Biotechnology, 2021, 39(3): 262-273.

[74] SAPOVAL N, AGHAZADEH A, NUTE MG, ANTUNES DA, BALAJI A, BARANIUK R, BARBERAN CJ, DANNENFELSER R, DUN C, EDRISI M, ELWORTH RAL, KILLE B, KYRILLIDIS A, NAKHLEH L, WOLFE CR, YAN Z, YAO V, TREANGEN TJ. Current progress and open challenges for applying deep learning across the biosciences[J]. Nature Communications, 2022, 13(1): 1728.

[75] SCHULER G, HU CY, KE AL. Structural basis for RNA-guided DNA cleavage by IscB-omega RNA and mechanistic comparison with Cas9[J]. Science, 2022, 376(6600): 1476-1481.

[76] AL-SHAYEB B, SKOPINTSEV P, SOCZEK KM, STAHL EC, LI Z, GROOVER E, SMOCK D, EGGERS AR, PAUSCH P, CRESS BF, HUANG CJ, STASKAWICZ B, SAVAGE DF, JACOBSEN SE, BANFIELD JF, DOUDNA JA. Diverse virus-encoded CRISPR-Cas systems include streamlined genome editors[J]. Cell, 2022, 185(24): 4574-4586.