Mining, engineering and functional expansion of CRISPR/Cas systems

doi:10.12211/2096-8280.2021-022

Abstract

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. {L-End}

Key words: CRISPR/Cas, gene editing, off-target effect, PAM constraints, Cas engineering

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。

关键词: CRISPR/Cas, 基因编辑, 脱靶效应, PAM约束, Cas工程改造

CLC Number:

Q816

Ke LIU, Guihong LIN, Kun LIU, Wei ZHOU, Fengqing WANG, Dongzhi WEI. 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.

Figures/Tables 7

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Cas蛋白变体	突变点	PAM序列	保真性	参考文献
KKH SaCas9	E782K/N968K/R1015H	NNNRRT	—	[75]
RRAsCas12a	S542R/K607R	TYCV	—	[74]
RVRAsCas12a	S542R/K548V/N552R	TATV	—	[74]
xCas9-3.7	A262T/R324L/S409I/E480K/E543D/M694I/E1219V	NG/NNG/GAA/GAT/CAA	提高	[77]
SpCas9-NRRH/NRTH/NRCH	—	NRNH	提高	[78]
HypaCas9	N692A/M694A/Q695A/H698A	NGG	提高	[79]
eSpCas9（1.0）	K810A/K1003A/R1060A	NGG	提高	[35]
SpCas9-HF1	N497A/R661A/Q695AQ926A	NGG	提高	[80]
SpG	D1135L/S1136W/G1218K/E1219Q/R1335Q/T1337R	NGN	—	[31]
SpRY	SpG突变点+L1111R/A1322R/R1333P/A61R/N1317R	NYN/NRN	—	[31]
enAsCas12a-HF1	E174R/S542R/K548R/N282A	TTYN/VTTV/TRTV	提高	[30]
LbCas12a-RVRR	G532R/K538V/Y542R/K595R	TNTN/TACV/TTCV/CTCV/CCCV	—	[38]
Blackjack SpCas9	—	NGG	提高	[37]

Cas蛋白变体	突变点	PAM序列	保真性	参考文献
KKH SaCas9	E782K/N968K/R1015H	NNNRRT	—	[75]
RRAsCas12a	S542R/K607R	TYCV	—	[74]
RVRAsCas12a	S542R/K548V/N552R	TATV	—	[74]
xCas9-3.7	A262T/R324L/S409I/E480K/E543D/M694I/E1219V	NG/NNG/GAA/GAT/CAA	提高	[77]
SpCas9-NRRH/NRTH/NRCH	—	NRNH	提高	[78]
HypaCas9	N692A/M694A/Q695A/H698A	NGG	提高	[79]
eSpCas9（1.0）	K810A/K1003A/R1060A	NGG	提高	[35]
SpCas9-HF1	N497A/R661A/Q695AQ926A	NGG	提高	[80]
SpG	D1135L/S1136W/G1218K/E1219Q/R1335Q/T1337R	NGN	—	[31]
SpRY	SpG突变点+L1111R/A1322R/R1333P/A61R/N1317R	NYN/NRN	—	[31]
enAsCas12a-HF1	E174R/S542R/K548R/N282A	TTYN/VTTV/TRTV	提高	[30]
LbCas12a-RVRR	G532R/K538V/Y542R/K595R	TNTN/TACV/TTCV/CTCV/CCCV	—	[38]
Blackjack SpCas9	—	NGG	提高	[37]

Cas蛋白	靶向核酸类型	CRISPR阵列加工	PAM/PFS	切割后的DNA末端	参考文献
Cas12b	dsDNA	无	富含T的PAM	黏性末端	[109]
Cas12c	dsDNA	无	—	—	[47]
Cas12d	dsDNA	无	富含T的PAM	—	[54]
Cas12e	dsDNA	无	富含T的PAM	—	[54]
CasФ	dsDNA	无	TBN	黏性末端	[48]
Cas12k	dsDNA	无	GTN	仅靶向不切割	[50]
Cas13b	ssRNA	是	5′D PFS 3′NAN/NNA	ssRNA以及邻近的RNA	[47]
Cas13c	ssRNA	无	—	—	[47]

Cas蛋白	靶向核酸类型	CRISPR阵列加工	PAM/PFS	切割后的DNA末端	参考文献
Cas12b	dsDNA	无	富含T的PAM	黏性末端	[109]
Cas12c	dsDNA	无	—	—	[47]
Cas12d	dsDNA	无	富含T的PAM	—	[54]
Cas12e	dsDNA	无	富含T的PAM	—	[54]
CasФ	dsDNA	无	TBN	黏性末端	[48]
Cas12k	dsDNA	无	GTN	仅靶向不切割	[50]
Cas13b	ssRNA	是	5′D PFS 3′NAN/NNA	ssRNA以及邻近的RNA	[47]
Cas13c	ssRNA	无	—	—	[47]

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