Optimization and development of CRISPR/Cas9 systems for genome editing

doi:10.12211/2096-8280.2022-047

Abstract

Abstract:

As an emerging technology developed within recent years, CRISPR/Cas9 exhibits fast, efficient, and precise gene editing and regulation capabilities in various organisms and tissues, and these advantages make it widely used in research with fundamental sciences and applied technologies as well such as synthetic biology. This review first briefly introduces the discovery history, classification, and mechanism of CRISPR/Cas9. The system of CRISPR/Cas9 usually contains a single guide RNA (gRNA) molecule for targeting a specific sequence, and a Cas9 endonuclease for catalyzing a double-strand break (DSB) in the sequence (target DNA strands). The recognition and cleavage of target DNA strictly require the presence of a protospacer adjacent motif (PAM) in the target sequence. The DSB(s) can be repaired by various DNA repair mechanisms, which allow various gene editing such as gene integration, gene replacement, and gene knockout. Due to limitations of CRISPR/Cas9, such as PAM dependence and high off-target rate, researchers have developed various fused or engineered Cas9 proteins and gRNAs that play significant roles in fulfilling various purposes. These Cas9 variants are modified for improving the performance of PAM, in particular its specificity and fidelity. Moreover, the DSBs generated by Cas9 are considered toxic to the cells, and the use of Cas9 nickase (nCas9) or catalytically deficient Cas9 (dCas9) in CRISPR has also been developed without generating DSBs. Meanwhile, different effector proteins can be fused with Cas9/dCas9/nCas9 to bring about new functions and applications in gene expression regulation, epigenome editing, and single base editing. Moreover, we introduce the current studies and applications of multiple gRNA expression strategies based on the multiplex advantages of the CRISPR/Cas9 system. In general, CRISPR/Cas9 systems have gradually become standardized and revolutionized genome editing systems for almost all possible genetic manipulations. Finally, we highlight perspectives on several applications of the versatile CRISPR/Cas9 toolbox as a genome editing tool, and discuss the safety and risk control issues when it is used in gene therapy. {L-End}

Key words: CRISPR/Cas9, genome editing, expression regulation, multiplexed genome editing, effector protein

摘要：

CRISPR/Cas9是近年来发展起来的新兴技术，其在多种生物和组织的基因组上具有快速、高效、精准的基因编辑与调控能力，这使得该技术在基础科学和合成生物学等应用科学领域均得到了极大的发展与应用。本文首先对CRISPR的历史沿革、分类及CRISPR/Cas9技术的作用机制进行简述，并结合其原理和在基因组工程中面临的脱靶率高、PAM依赖性强等限制因素总结了近年来针对Cas9蛋白和向导RNA（gRNA）进行的一系列优化与改造。接下来详细叙述了CRISPR/Cas9系统结合效应蛋白实现的多种功能，包括基因表达调控、表观基因组编辑、单碱基编辑等。基于gRNA多表达策略和Cas9多路复用策略，本文还对CRISPR/Cas9技术主要的多重应用成果进行了梳理汇总。最后探讨了CRISPR/Cas9作为高精度基因组编辑工具使用的应用前景，以及作为疗法使用时的安全性和风险控制问题。

关键词: CRISPR/Cas9, 基因组编辑, 表达调控, 多重编辑, 效应蛋白

CLC Number:

Q819

Xiaolong TENG, Shuobo SHI. Optimization and development of CRISPR/Cas9 systems for genome editing[J]. Synthetic Biology Journal, 2023, 4(1): 67-85.

滕小龙, 史硕博. CRISPR/Cas9系统在基因组编辑中的优化与发展[J]. 合成生物学, 2023, 4(1): 67-85.

Figures/Tables 6

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125	LIU K I, RAMLI M N B, WOO C W A, et al. A chemical-inducible CRISPR-Cas9 system for rapid control of genome editing[J]. Nature Chemical Biology, 2016, 12(11): 980-987.
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129	YEH W H, SHUBINA-OLEINIK O, LEVY J M, et al. In vivo base editing restores sensory transduction and transiently improves auditory function in a mouse model of recessive deafness[J]. Science Translational Medicine, 2020, 12(546): eaay9101.
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变体及直系同源	改造位点或方式	PAM	特性	参考文献
SpCas9	原生酿脓链球菌Cas9	NGG	1368个氨基酸	[29]
dCas9	D10A，H840A	NGG	核酸酶活性失活	[29]
nCas9(D10A)	D10A	NGG	切口酶，非靶向链切割活性失活	[30]
nCas9(H840A)	H840A	NGG	切口酶，靶向链切割活性失活	[30]
SpCas9NG	R1335V，L1111R，D1135V，G1218R，E1219F，A1322R，T1337R	NG	PAM改变；通过Cas9理性设计获得	[31]
VRERSpCas9	D1135A，G1218R，R1335E，T1337R	NGCG	PAM改变；通过细菌选择性定向进化获得	[19]
VQRSpCas9	D1135V，R1335Q，T1337R	NGAN，NGNG	PAM改变；通过细菌选择性定向进化获得	[19]
EQRSpCas9	D1335E，R1335Q，T1337R	NGAG	PAM改变；通过细菌选择性定向进化获得	[19]
xCas9	A262T，R324L，S409I，E480K，E543D，M694I，E1219V	NG，GAA，GAT	PAM改变；通过噬菌体辅助持续进化获得	[20]
SpG	D1135L，S1136W，G1218K，E1219Q，R1335Q，T1337R	NGN	PAM改变；通过SpCas9理性设计获得	[32]
SpRY	SpG，A61R，L1111R，A1322R，N1317R，R1333P	NRN，NYN	PAM改变；通过对SpG进一步理性设计获得	[32]
evoCas9	M495V, Y515N, K526E, R661Q	NGG	保真性提高，编辑效率接近天然SpCas9	[33]
fCas9	SpdCas9与FokI融合	NGG	保真性提高；通过将FokI核酸酶与dCas9融合获得	[34]
StCas9	原生嗜热链球菌Cas9	NNAGAAW	1121个氨基酸	[35]
NmCas9	原生脑膜炎奈瑟菌Cas9	NNNNGATT	1082个氨基酸	[36]
SaCas9	原生金黄色葡萄球菌Cas9	NNGRRT	1053个氨基酸；基因组编辑效率与SpCas9相当	[37]
CjCas9	原生曲状杆菌Cas9	NNNVRYM	984个氨基酸	[38]

变体及直系同源	改造位点或方式	PAM	特性	参考文献
SpCas9	原生酿脓链球菌Cas9	NGG	1368个氨基酸	[29]
dCas9	D10A，H840A	NGG	核酸酶活性失活	[29]
nCas9(D10A)	D10A	NGG	切口酶，非靶向链切割活性失活	[30]
nCas9(H840A)	H840A	NGG	切口酶，靶向链切割活性失活	[30]
SpCas9NG	R1335V，L1111R，D1135V，G1218R，E1219F，A1322R，T1337R	NG	PAM改变；通过Cas9理性设计获得	[31]
VRERSpCas9	D1135A，G1218R，R1335E，T1337R	NGCG	PAM改变；通过细菌选择性定向进化获得	[19]
VQRSpCas9	D1135V，R1335Q，T1337R	NGAN，NGNG	PAM改变；通过细菌选择性定向进化获得	[19]
EQRSpCas9	D1335E，R1335Q，T1337R	NGAG	PAM改变；通过细菌选择性定向进化获得	[19]
xCas9	A262T，R324L，S409I，E480K，E543D，M694I，E1219V	NG，GAA，GAT	PAM改变；通过噬菌体辅助持续进化获得	[20]
SpG	D1135L，S1136W，G1218K，E1219Q，R1335Q，T1337R	NGN	PAM改变；通过SpCas9理性设计获得	[32]
SpRY	SpG，A61R，L1111R，A1322R，N1317R，R1333P	NRN，NYN	PAM改变；通过对SpG进一步理性设计获得	[32]
evoCas9	M495V, Y515N, K526E, R661Q	NGG	保真性提高，编辑效率接近天然SpCas9	[33]
fCas9	SpdCas9与FokI融合	NGG	保真性提高；通过将FokI核酸酶与dCas9融合获得	[34]
StCas9	原生嗜热链球菌Cas9	NNAGAAW	1121个氨基酸	[35]
NmCas9	原生脑膜炎奈瑟菌Cas9	NNNNGATT	1082个氨基酸	[36]
SaCas9	原生金黄色葡萄球菌Cas9	NNGRRT	1053个氨基酸；基因组编辑效率与SpCas9相当	[37]
CjCas9	原生曲状杆菌Cas9	NNNVRYM	984个氨基酸	[38]

功能	效应蛋白或gRNA工程	系统	特征	参考文献
表达调控	无	dCas9	原核生物基因抑制表达，真核生物基因抑制效率低	[22]
	KRAB，MXI1，TUP1抑制结构域	dCas9	可在真核生物中抑制基因表达	[62-63]
	KRAB-MeCP1融合抑制结构域	dCas9	在哺乳动物细胞中高效抑制目的基因表达	[64]
	p65，VP64激活结构域	dCas9	激活基因表达，效率较低	[65-66]
	VP64-p65-Rta（VPR）融合激活结构域	dCas9	与VP64相比提高了22～320倍；多重基因激活表达；刺激iPSCs神经元分化	[67]
	gRNA工程；gRNA上携带携带两个MS2发夹二级结构	dCas9	每个发夹结构招募一个MS2-p65-HSF1融合激活蛋白；与VP64相比，使10个靶基因上调2倍以上	[68]
	SPY系统（包括SpyTag和SpyCatcher）和Med2激活结构域	dCas9	招募多拷贝SpyCatcher和Med2融合蛋白使酿酒酵母目的基因表达提高34.9倍	[69]
	SunTag系统（包括GCN4肽和抗GCN4抗体的单链可变片段scFv）和MIG1抑制结构域	dCpf1	招募多拷贝ScFv与MIG1融合蛋白使酿酒酵母目的基因受到95%的抑制	[69]
表观基因组编辑	KRAB-DNA甲基化酶催化结构域融合蛋白	dCas9	融合抑制因子和甲基化酶；造成目的基因78%的稳定沉默	[70]
	两侧分别融合KRAB和Dnmt3A-Dnmt3L融合DNA甲基化酶结构域	dCas9	在干细胞分裂分化为神经细胞后仍维持DNA甲基化和基因抑制	[71]
	TET1甲基胞嘧啶双加氧酶1催化结构域	dCas9	dCas9- TET1靶向至BRCA1基因启动子后成功地上调其表达水平	[72]
	人类组蛋白乙酰基转移酶p300催化核心	dCas9	催化组蛋白H3 第27位赖氨酸残基的乙酰基化；基因表达上调	[73]
单碱基编辑	胞嘧啶脱氨酶，腺嘌呤脱氨酶	nCas9	在不造成DNA双链断裂的条件下使目的碱基产生C:G与T:A或A:T与G:C的碱基对替换	[30,74-75]
	CBE-UGI融合蛋白	nCas9	UGI能够防止胞嘧啶C脱氨后的尿嘧啶U被切除，从而显著提高了CBEs的编辑效率	[30]
	单链DNA结合结构域ssDBD融合到胞嘧啶脱氨酶与nCas9之间	nCas9	通过延长ssDNA的暴露时间极大地提高CBEs的编辑效率；使CBEs的编辑窗口范围得到了提高	[76]
	组合nCas9、胞嘧啶脱氨酶和尿嘧啶-DNA糖基化酶	nCas9	首次实现大肠杆菌中C到A 87.2%和哺乳动物细胞C到G 5.3%至53.0%的编辑效率	[77]
先导编辑	通过gRNA工程改造的pegRNA；与逆转录酶融合	nCas9	改变pegRNA可实现精确的碱基替换、基因敲除和插入	[27]
	通过gRNA工程改造的pegRNA；与逆转录酶和Rad51融合	nCas9	融合单链DNA结合蛋白Rad51使PE2的编辑效率最高提升2.6倍	[78]

功能	效应蛋白或gRNA工程	系统	特征	参考文献
表达调控	无	dCas9	原核生物基因抑制表达，真核生物基因抑制效率低	[22]
	KRAB，MXI1，TUP1抑制结构域	dCas9	可在真核生物中抑制基因表达	[62-63]
	KRAB-MeCP1融合抑制结构域	dCas9	在哺乳动物细胞中高效抑制目的基因表达	[64]
	p65，VP64激活结构域	dCas9	激活基因表达，效率较低	[65-66]
	VP64-p65-Rta（VPR）融合激活结构域	dCas9	与VP64相比提高了22～320倍；多重基因激活表达；刺激iPSCs神经元分化	[67]
	gRNA工程；gRNA上携带携带两个MS2发夹二级结构	dCas9	每个发夹结构招募一个MS2-p65-HSF1融合激活蛋白；与VP64相比，使10个靶基因上调2倍以上	[68]
	SPY系统（包括SpyTag和SpyCatcher）和Med2激活结构域	dCas9	招募多拷贝SpyCatcher和Med2融合蛋白使酿酒酵母目的基因表达提高34.9倍	[69]
	SunTag系统（包括GCN4肽和抗GCN4抗体的单链可变片段scFv）和MIG1抑制结构域	dCpf1	招募多拷贝ScFv与MIG1融合蛋白使酿酒酵母目的基因受到95%的抑制	[69]
表观基因组编辑	KRAB-DNA甲基化酶催化结构域融合蛋白	dCas9	融合抑制因子和甲基化酶；造成目的基因78%的稳定沉默	[70]
	两侧分别融合KRAB和Dnmt3A-Dnmt3L融合DNA甲基化酶结构域	dCas9	在干细胞分裂分化为神经细胞后仍维持DNA甲基化和基因抑制	[71]
	TET1甲基胞嘧啶双加氧酶1催化结构域	dCas9	dCas9- TET1靶向至BRCA1基因启动子后成功地上调其表达水平	[72]
	人类组蛋白乙酰基转移酶p300催化核心	dCas9	催化组蛋白H3 第27位赖氨酸残基的乙酰基化；基因表达上调	[73]
单碱基编辑	胞嘧啶脱氨酶，腺嘌呤脱氨酶	nCas9	在不造成DNA双链断裂的条件下使目的碱基产生C:G与T:A或A:T与G:C的碱基对替换	[30,74-75]
	CBE-UGI融合蛋白	nCas9	UGI能够防止胞嘧啶C脱氨后的尿嘧啶U被切除，从而显著提高了CBEs的编辑效率	[30]
	单链DNA结合结构域ssDBD融合到胞嘧啶脱氨酶与nCas9之间	nCas9	通过延长ssDNA的暴露时间极大地提高CBEs的编辑效率；使CBEs的编辑窗口范围得到了提高	[76]
	组合nCas9、胞嘧啶脱氨酶和尿嘧啶-DNA糖基化酶	nCas9	首次实现大肠杆菌中C到A 87.2%和哺乳动物细胞C到G 5.3%至53.0%的编辑效率	[77]
先导编辑	通过gRNA工程改造的pegRNA；与逆转录酶融合	nCas9	改变pegRNA可实现精确的碱基替换、基因敲除和插入	[27]
	通过gRNA工程改造的pegRNA；与逆转录酶和Rad51融合	nCas9	融合单链DNA结合蛋白Rad51使PE2的编辑效率最高提升2.6倍	[78]

宿主细胞	多重CRISPR策略	靶向目标数量	应用/概念验证	参考文献
哺乳动物细胞	2个间隔区通过天然CRISPR序列自我加工	2个靶标(1个基因)	对1个靶基因敲除，效率为1.6%	[4]
酿酒酵母	Casy4核酸酶加工	3个基因	同时对3个不同启动子进行激活，使得3个报告基因荧光强度提升了2倍	[113]
酿酒酵母	基于Csy4核酸酶加工的多gRNA快速组装	12个靶标(3个基因)	通过12个gRNA靶向使得3个报告基因荧光强度分别被抑制了92%、81%和95%	[114]
酿酒酵母	基于tRNA转录本切割机制的多gRNA表达系统	8个基因	通过两轮8个基因的敲除获得了30倍的游离脂肪酸产量	[115]
酿酒酵母	多个gRNA表达盒通过pol Ⅲ启动子启动	3个基因	将适配子与gRNA融合进行多基因调控，获得了不同的紫罗兰素生物合成产物	[118]
酿酒酵母	基于Csy4核酸酶加工的多gRNA快速组装策略及多Cas9正交组合调控	3个基因	通过组合调控使两个报告基因表达分别被抑制和激活5倍，同时以95%的效率敲除第三个基因	[119]
酿酒酵母	通过质粒表达多个gRNA表达盒以及截短gRNA用于调控	3个基因	通过对三个基因分别进行编辑、激活和抑制获得了α-檀香烯2.66倍的产量	[43]
马克思克鲁维酵母(Kluyveromyces marxianus)	基于tRNA转录本切割机制的多gRNA表达系统	6个靶标(4个基因)	通过对4个基因进行调控，使乙酸乙酯的产量提升了3.8倍	[120]
大肠杆菌	多个gRNA表达盒通过质粒表达	3个基因	通过对3个基因进行组合调控得到了2.3倍苹果酸产量菌株	[121]
大肠杆菌	多个gRNA表达盒通过质粒表达	3个基因	对苹果酸合成途径基因进行组合调控后获得了2.3倍苹果酸产量菌株	[121]
枯草芽孢杆菌(Bacillus subtilis)	多个gRNA表达盒整合至基因组	3个基因	通过多基因动态组合调控提高了N-乙酰葡糖胺产量	[122]
天蓝色链霉菌(Streptomyces coelicolor)	多个gRNA表达盒与dCas9在质粒上表达	4个基因	通过对4个靶基因同时进行抑制，使其mRNA表达量降为对照的2%～32%	[123]