Mining, engineering and functional expansion of CRISPR/Cas systems

LIU Ke; LIN Guihong; LIU Kun; ZHOU Wei; WANG Fengqing; WEI Dongzhi

doi:10.12211/2096-8280.2021-022

您当前的位置：

首页 >

文章列表页 >

Mining, engineering and functional expansion of CRISPR/Cas systems

Invited Review | 更新时间：2025-03-20

- Mining, engineering and functional expansion of CRISPR/Cas systems
- Synthetic Biology Journal Vol. 4, Issue 1, Pages: 47-66(2023)
- 作者机构：
  
  华东理工大学生物反应器工程国家重点实验室，鲁华生物技术研究所，上海 200237
- 作者简介：
- 基金信息：
- DOI：10.12211/2096-8280.2021-022
  CLC： Q816
- Received：08 February 2021，
  
  Revised：2021-04-28，
  
  Published：28 February 2023
- 稿件说明：
移动端阅览
柳柯，林桂虹，刘坤，周伟，王风清，魏东芝. CRISPR/Cas系统的挖掘、改造与功能拓展［J］. 合成生物学， 2023， 4（1）： 47-66

LIU Ke， LIN Guihong， LIU Kun， ZHOU Wei， WANG Fengqing， WEI Dongzhi. Mining， engineering and functional expansion of CRISPR/Cas systems［J］. Synthetic Biology Journal， 2023， 4（1）： 47-66
柳柯，林桂虹，刘坤，周伟，王风清，魏东芝. CRISPR/Cas系统的挖掘、改造与功能拓展［J］. 合成生物学， 2023， 4（1）： 47-66 DOI： 10.12211/2096-8280.2021-022.

LIU Ke， LIN Guihong， LIU Kun， ZHOU Wei， WANG Fengqing， WEI Dongzhi. Mining， engineering and functional expansion of CRISPR/Cas systems［J］. Synthetic Biology Journal， 2023， 4（1）： 47-66 DOI： 10.12211/2096-8280.2021-022.

摘要

规律成簇间隔短回文重复序列及其相关蛋白（CRISPR/Cas）是一种微生物获得性免疫系统，自从证实其可用于基因编辑之后，迅速增强了我们编辑、操纵、注释、检测甚至成像生物体DNA和RNA的能力，为基础生命科学、医学和生物工程等领域的创新发展注入了强劲动力，快速推动了合成生物学等学科的兴盛发展。然而，CRISPR/Cas系统也有一些固有的问题，例如脱靶效应、原间隔序列邻近基序（PAM）对靶目标的约束性以及基因编辑活性的可控性等，严重制约了该系统在基因精准可控编辑等方面的长足发展，阻碍了其新功能和新应用的拓展。为了突破这些限制，“蛋白质工程修饰Cas蛋白”与“基于生物信息学的新型CRISPR/Cas系统的挖掘”就成为完善发展CRISPR/Cas系统以及扩充CRISPR工具箱的两种重要策略。本文主要针对当前应用最为广泛的Ⅱ类CRISPR/Cas系统，重点介绍了CRISPR/Cas9、CRISPR/Cas12a和CRISPR/Cas13a这三种代表性系统的基本结构和作用机制，及其在结构改造和功能拓展等方面的新进展，同时也对一些新近发掘的具有重要特色和潜在应用价值的CRISPR/Cas系统进行了综述，例如CRISPR/CasФ 和 CRISPR/Cas12k。这些改造和发掘工作显著改善了CRISPR/Cas系统的固有问题，有力地拓展了其功能和适用性，势必会进一步快速推动CRISPR/Cas系统在诸多领域的创新发展。

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) were derived from an acquired immune system in microbes. Since their functions on gene editing have been reported

they have been rapidly used to enhance our ability to edit

regulate

annotate

detect

and image DNA and RNA fragments of various organisms

which consequently have faciliated fundamental research in life science

medicine

bioengineering and so on

and drived the development of synthetic biology and other disciplines. However

CRISPR/Cas systems also have some inherent drawbacks

such as off-target effect

constraint of protospacer-adjacent motif (PAM) on the target

and controllability of the gene editing activity

which substantially compromise their applications in highly precise and controllable gene editing. In order to overcome these challenges

two important strategies have been employed to develop enhanced CRISPR/Cas systems and expand the CRISPR toolbox

including modifying the Cas proteins by protein engineering and mining novel CRISPR/Cas systems with bioinformatics. In the review

focusing on the most widely used the type II CRISPR/Cas systems

we mainly introduce the basic structures and functions of three representative systems

including CRISPR/Cas9

CRISPR/Cas12a and CRISPR/Cas13a

as well as recent progress in their structural modifications and functional expansion. Thereinto

engineering strategies for CRISPR/Cas systems have been systematically commented

which include modification methods for Cas proteins and the way to expand the function of CRISPR/Cas systems by coupling specfic proteins with Cas. In addition

we also review some novel CRISPR/Cas systems with important characteristics and potential applications that have been discovered in recently years

such as CRISPR/CasФ and CRISPR/Cas12k. These engineering modifications and mining work have greatly addressed the inherent problems of the CRISPR/Cas systems

and effectively expanded their functions and applicability

which will further promote the applications of the CRISPR/Cas systems in many fields.

关键词

Keywords

references

MARRAFFINI L A . CRISPR-Cas immunity in prokaryotes [J ] . Nature , 2015 , 526 ( 7571 ): 55 - 61 .

GARNEAU J E , DUPUIS M-È , VILLION M , et al . The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA [J ] . Nature , 2010 , 468 ( 7320 ): 67 - 71 .

TERNS M P , TERNS R M . CRISPR-based adaptive immune systems [J ] . Current Opinion in Microbiology , 2011 , 14 ( 3 ): 321 - 327 .

AMITAI G , SOREK R . CRISPR-Cas adaptation: Insights into the mechanism of action [J ] . Nature Reviews Microbiology , 2016 , 14 ( 2 ): 67 - 76 .

CHARPENTIER E , RICHTER H , VAN DER OOST J , et al . Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity [J ] . FEMS Microbiology Reviews , 2015 , 39 ( 3 ): 428 - 441 .

PLAGENS A , RICHTER H , CHARPENTIER E , et al . DNA and RNA interference mechanisms by CRISPR-Cas surveillance complexes [J ] . FEMS Microbiology Reviews , 2015 , 39 ( 3 ): 442 - 463 .

NISHIMASU H , NUREKI O . Structures and mechanisms of CRISPR RNA-guided effector nucleases [J ] . Current Opinion in Structural Biology , 2017 , 43 : 68 - 78 .

MILLER J C , HOLMES M C , WANG J B , et al . An improved zinc-finger nuclease architecture for highly specific genome editing [J ] . Nature Biotechnology , 2007 , 25 ( 7 ): 778 - 785 .

CERMAK T , DOYLE E L , CHRISTIAN M , et al . Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting [J ] . Nucleic Acids Research , 2011 , 39 ( 12 ): e82 .

SANTOS-MORENO J , TASIUDI E , STELLING J , et al . Multistable and dynamic CRISPRi-based synthetic circuits [J ] . Nature Communications , 2020 , 11 : 2746 .

LIU Y , WAN X Y , WANG B J . Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria [J ] . Nature Communications , 2019 , 10 : 3693 .

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 .

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 .

THAKORE P I , BLACK J B , HILTON I B , et al . Editing the epigenome: technologies for programmable transcription and epigenetic modulation [J ] . Nature Methods , 2016 , 13 ( 2 ): 127 - 137 .

HILTON I B , D′IPPOLITO A M , VOCKLEY C M , et al . Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers [J ] . Nature Biotechnology , 2015 , 33 ( 5 ): 510 - 517 .

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 .

NISHIDA K , ARAZOE T , YACHIE N , et al . Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems [J ] . Science , 2016 , 353 ( 6305 ): aaf8729 .

DREISSIG S , SCHIML S , SCHINDELE P , et al . Live-cell CRISPR imaging in plants reveals dynamic telomere movements [J ] . The Plant Journal: for Cell and Molecular Biology , 2017 , 91 ( 4 ): 565 - 573 .

ROUET R , THUMA B A , ROY M D , et al . Receptor-mediated delivery of CRISPR-Cas9 endonuclease for cell-type-specific gene editing [J ] . Journal of the American Chemical Society , 2018 , 140 ( 21 ): 6596 - 6603 .

ZHOU Y X , WANG P , TIAN F , et al . Painting a specific chromosome with CRISPR/Cas9 for live-cell imaging [J ] . Cell Research , 2017 , 27 ( 2 ): 298 - 301 .

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

BAO Z H , XIAO H , LIANG J , et al . Homology-integrated CRISPR-Cas (HI-CRISP R) system for one-step multigene disruption in Saccharomyces cerevisiae [J ] . ACS Synthetic Biology , 2015 , 4 ( 5 ): 585 - 594 .

XIE K B , YANG Y N . RNA-guided genome editing in plants using a CRISPR-Cas system [J ] . Molecular Plant , 2013 , 6 ( 6 ): 1975 - 1983 .

GUAN L H , HAN Y W , ZHU S Y , et al . Application of CRISPR-Cas system in gene therapy: pre-clinical progress in animal model [J ] . DNA Repair , 2016 , 46 : 1 - 8 .

MODARAI S R , KANDA S , BLOH K , et al . Precise and error-prone CRISPR-directed gene editing activity in human CD34+ cells varies widely among patient samples [J ] . Gene Therapy , 2021 , 28 ( 1/2 ): 105 - 113 .

MARX V . Guide RNAs: It's good to be choosy [J ] . Nature Methods , 2020 , 17 ( 12 ): 1179 - 1182 .

FU Y F , FODEN J A , KHAYTER C , et al . High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells [J ] . Nature Biotechnology , 2013 , 31 ( 9 ): 822 - 826 .

TASAN I , ZHAO H M . Targeting specificity of the CRISPR/Cas9 system [J ] . ACS Synthetic Biology , 2017 , 6 ( 9 ): 1609 - 1613 .

WANG Y , WANG M R , ZHENG T , et al . Specificity profiling of CRISPR system reveals greatly enhanced off-target gene editing [J ] . Scientific Reports , 2020 , 10 ( 1 ): 2269 .

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 .

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 .

GILBERT L A , HORLBECK M A , ADAMSON B , et al . Genome-scale CRISPR-mediated control of gene repression and activation [J ] . Cell , 2014 , 159 ( 3 ): 647 - 661 .

CHO S W , KIM S , KIM Y , et al . Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases [J ] . Genome Research , 2014 , 24 ( 1 ): 132 - 141 .

ZETSCHE B , VOLZ S E , ZHANG F . A split-Cas9 architecture for inducible genome editing and transcription modulation [J ] . Nature Biotechnology , 2015 , 33 ( 2 ): 139 - 142 .

SLAYMAKER I M , GAO L Y , ZETSCHE B , et al . Rationally engineered Cas9 nucleases with improved specificity [J ] . Science , 2016 , 351 ( 6268 ): 84 - 88 .

GAO Z L , HERRERA-CARRILLO E , BERKHOUT B . A single H1 promoter can drive both guide RNA and endonuclease expression in the CRISPR-Cas9 system [J ] . Molecular Therapy-Nucleic Acids , 2019 , 14 : 32 - 40 .

KULCSÁR P I , TÁLAS A , TÓTH E , et al . Blackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5′G-extended sgRNAs [J ] . Nature Communications , 2020 , 11 ( 1 ): 1223 .

TÓTH E , VARGA É , KULCSÁR P I , et al . Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases [J ] . Nucleic Acids Research , 2020 , 48 ( 7 ): 3722 - 3733 .

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 .

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 .

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 .

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 .

LIU R M , LIANG L L , FREED E , et al . Synthetic chimeric nucleases function for efficient genome editing [J ] . Nature Communications , 2019 , 10 : 5524 .

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 .

HIRANO H , GOOTENBERG J S , HORII T , et al . Structure and engineering of francisella novicida Cas9 [J ] . Cell , 2016 , 164 ( 5 ): 950 - 961 .

SHMAKOV S , ABUDAYYEH O O , MAKAROVA K S , et al . Discovery and functional characterization of diverse class 2 CRISPR-Cas systems [J ] . Molecular Cell , 2015 , 60 ( 3 ): 385 - 397 .

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 .

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 .

MOHANRAJU P , MAKAROVA K S , ZETSCHE B , et al . Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems [J ] . Science , 2016 , 353 ( 6299 ): aad5147 .

STRECKER J , LADHA A , GARDNER Z , et al . RNA-guided DNA insertion with CRISPR-associated transposases [J ] . Science , 2019 , 365 ( 6448 ): 48 - 53 .

LIU L , CHEN P , WANG M , et al . C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism [J ] . Molecular Cell , 2017 , 65 ( 2 ): 310 - 322 .

HARRINGTON L B , MA E B , CHEN J S , et al . A scoutRNA is required for some type V CRISPR-Cas systems [J ] . Molecular Cell , 2020 , 79 ( 3 ): 416 - 424.e5 .

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

BURSTEIN D , HARRINGTON L B , STRUTT S C , et al . New CRISPR-Cas systems from uncultivated microbes [J ] . Nature , 2017 , 542 ( 7640 ): 237 - 241 .

MAKAROVA K S , WOLF Y I , ALKHNBASHI O S , et al . An updated evolutionary classification of CRISPR-Cas systems [J ] . Nature Reviews Microbiology , 2015 , 13 ( 11 ): 722 - 736 .

MAKAROVA K S , KOONIN E V . Annotation and classification of CRISPR-Cas systems [M ] //LUNDGREN M, CHARPENTIER E, FINERAN P.CRISPR. Methods in Molecular Biology . New York : Humana Press , 2015 , 1311 : 47 - 75 .

HOCHSTRASSER M L , TAYLOR D W , BHAT P , et al . CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference [J ] . Proceedings of the National Academy of Sciences of the United States of America , 2014 , 111 ( 18 ): 6618 - 6623 .

BROUNS S J J , JORE M M , LUNDGREN M , et al . Small CRISPR RNAs guide antiviral defense in prokaryotes [J ] . Science , 2008 , 321 ( 5891 ): 960 - 964 .

JINEK M , CHYLINSKI K , FONFARA I , et al . A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity [J ] . Science , 2012 , 337 ( 6096 ): 816 - 821 .

CONG L , RAN F A , COX D , et al . Multiplex genome engineering using CRISPR/Cas systems [J ] . Science , 2013 , 339 ( 6121 ): 819 - 823 .

JIANG F G , DOUDNA J A . CRISPR-Cas9 structures and mechanisms [J ] . Annual Review of Biophysics , 2017 , 46 : 505 - 529 .

STERNBERG S H , LAFRANCE B , KAPLAN M , et al . Conformational control of DNA target cleavage by CRISPR-Cas9 [J ] . Nature , 2015 , 527 ( 7576 ): 110 - 113 .

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 .

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 .

JASIN M , HABER J E . The democratization of gene editing: insights from site-specific cleavage and double-strand break repair [J ] . DNA Repair , 2016 , 44 : 6 - 16 .

BÉTERMIER M , BERTRAND P , LOPEZ B S . Is non-homologous end-joining really an inherently error-prone process? [J ] . PLoS genetics , 2014 , 10 ( 1 ): e1004086 .

NAKADE S , TSUBOTA T , SAKANE Y , et al . Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9 [J ] . Nature Communications , 2014 , 5 : 5560 .

SUZUKI K , TSUNEKAWA Y , HERNANDEZ-BENITEZ R , et al . In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration [J ] . Nature , 2016 , 540 ( 7631 ): 144 - 149 .

HSU P D , LANDER E S , ZHANG F . Development and applications of CRISPR-Cas9 for genome engineering [J ] . Cell , 2014 , 157 ( 6 ): 1262 - 1278 .

CHAMBERLAIN K , RIYAD J M , WEBER T . Expressing transgenes that exceed the packaging capacity of adeno-associated virus capsids [J ] . Human Gene Therapy Methods , 2016 , 27 ( 1 ): 1 - 12 .

YANG S , LI S H , LI X J . Shortening the half-life of Cas9 maintains its gene editing ability and reduces neuronal toxicity [J ] . Cell Reports , 2018 , 25 ( 10 ): 2653 - 2659 .

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 .

LIANG M D , LI Z L , WANG W S , et al . A CRISPR-Cas12a-derived biosensing platform for the highly sensitive detection of diverse small molecules [J ] . Nature Communications , 2019 , 10 : 3672 .

GIER R A , BUDINICH K A , EVITT N H , et al . High-performance CRISPR-Cas12a genome editing for combinatorial genetic screening [J ] . Nature Communications , 2020 , 11 : 3455 .

YAN M Y , YAN H Q , REN G X , et al . CRISPR-Cas12a-assisted recombineering in bacteria [J ] . Applied and Environmental Microbiology , 2017 , 83 ( 17 ): e00947-17 .

TANG X , LOWDER L G , ZHANG T , et al . A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants [J ] . Nature Plants , 2017 , 3 ( 3 ): 17018 .

VERWAAL R , BUITING-WIESSENHAAN N , DALHUIJSEN S , et al . CRISPR/Cpf1 enables fast and simple genome editing of Saccharomyces cerevisiae [J ] . Yeast , 2018 , 35 ( 2 ): 201 - 211 .

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 .

STELLA S , ALCÓN P , MONTOYA G . Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage [J ] . Nature , 2017 , 546 ( 7659 ): 559 - 563 .

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 .

ABUDAYYEH O O , GOOTENBERG J S , ESSLETZBICHLER P , et al . RNA targeting with CRISPR-Cas13 [J ] . Nature , 2017 , 550 ( 7675 ): 280 - 284 .

KNOTT G J , EAST-SELETSKY A , COFSKY J C , et al . Guide-bound structures of an RNA-targeting A-cleaving CRISPR-Cas13a enzyme [J ] . Nature Structural & Molecular Biology , 2017 , 24 ( 10 ): 825 - 833 .

ABUDAYYEH O O , GOOTENBERG J S , KONERMANN S , et al . C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector [J ] . Science , 2016 , 353 ( 6299 ): aaf5573 .

LIU L , LI X Y , MA J , et al . The molecular architecture for RNA-guided RNA cleavage by Cas13a [J ] . Cell , 2017 , 170 ( 4 ): 714 - 726.e10 .

GOOTENBERG J S , ABUDAYYEH O O , LEE J W , et al . Nucleic acid detection with CRISPR-Cas13a/C2c2 [J ] . Science , 2017 , 356 ( 6336 ): 438 - 442 .

FOZOUNI P , SON S M , DE LEÓN DERBY M D , et al . Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy [J ] . Cell , 2021 , 184 ( 2 ): 323 - 333.e9 .

PYZOCHA N K , CHEN S D . Diverse class 2 CRISPR-Cas effector proteins for genome engineering applications [J ] . ACS Chemical Biology , 2018 , 13 ( 2 ): 347 - 356 .

KIM D , KIM J , HUR J K , et al . Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells [J ] . Nature Biotechnology , 2016 , 34 ( 8 ): 863 - 868 .

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 .

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 .

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 .

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 .

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 .

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 .

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 .

KIM N , KIM H K , LEE S , et al . Prediction of the sequence-specific cleavage activity of Cas9 variants [J ] . Nature Biotechnology , 2020 , 38 ( 11 ): 1328 - 1336 .

ZHOU X X , ZOU X Z , CHUNG H K , et al . A single-chain photoswitchable CRISPR-Cas9 architecture for light-inducible gene editing and transcription [J ] . ACS Chemical Biology , 2018 , 13 ( 2 ): 443 - 448 .

WANG Y , CHENG H J , LIU Y , et al . In-situ generation of large numbers of genetic combinations for metabolic reprogramming via CRISPR-guided base editing [J ] . Nature Communications , 2021 , 12 : 678 .

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 .

GE R Q , MAI G Q , WANG P , et al . CRISPRdigger: detecting CRISPRs with better direct repeat annotations [J ] . Scientific Reports , 2016 , 6 : 32942 .

GRISSA I , VERGNAUD G , POURCEL C . CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats [J ] . Nucleic Acids Research , 2007 , 35 ( suppl_2 ): W52 - W57 .

BLAND C , RAMSEY T L , SABREE F , et al . CRISPR recognition tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats [J ] . BMC Bioinformatics , 2007 , 8 : 209 .

ABBY S S , NÉRON B , MÉNAGER H , et al . MacSyfinder: a program to mine genomes for molecular systems with an application to CRISPR-Cas systems [J ] . PLoS One , 2014 , 9 ( 10 ): e110726 .

CHAI G S , YU M , JIANG L X , et al . HMMCAS: a web tool for the identification and domain annotations of Cas proteins [J ] . IEEE/ACM Transactions on Computational Biology and Bioinformatics , 2019 , 16 ( 4 ): 1313 - 1315 .

COUVIN D , BERNHEIM A , TOFFANO-NIOCHE C , et al . CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins [J ] . Nucleic Acids Research , 2018 , 46 ( W1 ): W246 - W251 .

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 .

CUI Y , TANG Y C , LIANG M , et al . Direct observation of the formation of CRISPR-Cas12a R-loop complex at the single-molecule level [J ] . Chemical Communications , 2020 , 56 ( 14 ): 2123 - 2126 .

KLOMPE S E , VO P L H , HALPIN-HEALY T S , et al . Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration [J ] . Nature , 2019 , 571 ( 7764 ): 219 - 225 .

YANG H , GAO P , RAJASHANKAR K R , et al . PAM-dependent target DNA recognition and cleavage by C2c1 CRISPR-Cas endonuclease [J ] . Cell , 2016 , 167 ( 7 ): 1814 - 1828.e12 .

SHIN J , OH J W . Development of CRISPR/Cas9 system for targeted DNA modifications and recent improvements in modification efficiency and specificity [J ] . BMB Reports , 2020 , 53 ( 7 ): 341 - 348 .

ZHENG T , HOU Y Z , ZHANG P J , et al . Profiling single-guide RNA specificity reveals a mismatch sensitive core sequence [J ] . Scientific Reports , 2017 , 7 : 40638 .

KIGA K , TAN X E , IBARRA-CHÁVEZ R , et al . Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria [J ] . Nature Communications , 2020 , 11 ( 1 ): 2934 .

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Biosafety strategies for engineered bacteria

Applications of synthetic biology in medical diagnosis and treatment： technological breakthroughs and ethical issues

Progress and challenges of synthetic biology in agriculture

Related Author

Ke LIU

Guihong LIN

Kun LIU

Wei ZHOU

Fengqing WANG

Dongzhi WEI

CHEN Yingying

LIU Yang

Related Institution

State Key Laboratory of Bioreactor Engineering， Newworld Institute of Biotechnology， East China University of Science and Technology

School of Pharmaceutical Sciences， Key Laboratory of Chemical Biology （Ministry of Education）， Shandong Basic Science Research Center （Pharmacy）， Key Laboratory of Medicinal Chemical Biology （Shandong）， Shandong University

CAS Key Laboratory of Tropical Marine Bio-resources and Ecology， Guangdong Key Laboratory of Marine Materia Medica， South China Sea Institute of Oceanology， Chinese Academy of Sciences

Functional Nano and Soft Materials （FUNSOM）， Soochow University

Institute of Synthetic Biology， Shenzhen Institutes of Advanced Technology， Chinese Academy of Sciences

⁰