CRISPR-Cas基因编辑系统升级：聚焦Cas蛋白和PAM

doi:10.16288/j.yczz.19-297

遗传 ›› 2020, Vol. 42 ›› Issue (3): 236-249.doi: 10.16288/j.yczz.19-297

CRISPR-Cas基因编辑系统升级：聚焦Cas蛋白和PAM

唐连超, 谷峰()

温州医科大学附属眼视光医院,眼视光学和视觉科学国家重点实验室,温州 325027

收稿日期:2019-11-12 修回日期:2020-02-26 出版日期:2020-03-20 发布日期:2020-03-04
通讯作者: 谷峰 E-mail:fgu@mail.eye.ac.cn
作者简介:唐连超,硕士研究生,专业方向：基因编辑工具优化及应用。E-mail: 1664089980@qq.com
基金资助:
国家自然科学基金项目资助编号(81201181)

Next-generation CRISPR-Cas for genome editing: focusing on the Cas protein and PAM

Lianchao Tang, Feng Gu()

State Key Laboratory of Ophthalmology and Optometry, School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou 325027, China

Received:2019-11-12 Revised:2020-02-26 Online:2020-03-20 Published:2020-03-04
Contact: Gu Feng E-mail:fgu@mail.eye.ac.cn
Supported by:
Supported by the National Natural Science Foundation of China No(81201181)

摘要/Abstract

摘要：

以CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR associated proteins)系统为代表的基因编辑技术的出现极大地促进了人类改造自然界物种的能力。在医疗、工业、农业等多个研究领域,基因编辑技术正在被广泛应用。Cas蛋白是CRISPR-Cas系统的功能蛋白,不同类型的Cas蛋白在其自身活性、识别位点、切割末端、RNA需求等方面具有不同的特性。PAM (protospacer adjacent motif)是靶位点附近的若干个碱基,对Cas蛋白识别靶序列至关重要,也是CRISPR-Cas系统发挥功效的关键特性之一。目前已有多种不同的PAM鉴定方法被报道。本文对Cas蛋白的寻找、Cas蛋白突变体筛选及PAM的确定方法(含PAM谱拓展)进行了综述,以期为新型基因编辑工具的发展和优化提供借鉴。

关键词: 基因编辑, CRISPR-Cas系统, Cas蛋白, PAM, 定向进化

Abstract:

The emergence of the gene editing technology, especial CRISPR-Cas (clustered regularly intersected short palindromic repeats and CRISPR associated proteins), has greatly promoted the ability of human beings to transform natural species. It has been widely harnessed for the engineering in the medical, industrial, agricultural and other fields. The key component of the CRISPR-Cas system, the Cas protein, possesses its specific features, including self-activity, recognition site, cutting end and guide RNA. PAM (protein assistant motif) is a number of nucleotides adjacent to the target site, which is very important for the Cas protein to recognize the target sequence and is also the key characteristic of CRISPR-Cas. There are several reported methods for identification of PAM. In this review, we summarize the searching for the Cas protein, the identification of Cas mutants with desired traits and the mapping of the PAM (including the extending of PAM spectrum), in order to provide a reference for the development and optimization of next-generation gene editing tools.

Key words: gene editing, CRISPR-Cas system, Cas protein, PAM, directed evolution

唐连超, 谷峰. CRISPR-Cas基因编辑系统升级：聚焦Cas蛋白和PAM[J]. 遗传, 2020, 42(3): 236-249.

Lianchao Tang, Feng Gu. Next-generation CRISPR-Cas for genome editing: focusing on the Cas protein and PAM[J]. Hereditas(Beijing), 2020, 42(3): 236-249.

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方法	特点	劣势	Cas蛋白	文献
生物信息学分析	一次能够发现大量结构相似或者功能类似的蛋白; 不需要进行分子实验	需要已知的序列信息	SpCas9	[16]
			SaCas9	[25]
			Cpf1	[11]
			FnCas9	[59]
			NmCas9	[60]
			CjCas9	[61]
			St1Cas9	[40]
			St3Cas9	[40]
基于质粒干扰的耗尽筛选	实验思路简单,将PAM识别与细菌的死亡相偶联	通过盒式诱变制作Cas蛋白突变库, 工作量较大; 需要进行第二代测序	AsCpf1 RR	[33]
基于质粒干扰的耗尽筛选	实验思路简单,将PAM识别与细菌的死亡相偶联	通过盒式诱变制作Cas蛋白突变库, 工作量较大; 需要进行第二代测序	AsCpf1 RVR	[33]
细菌选择性定向进化	将PAM识别与细菌的生存相偶联	需制作Cas蛋白突变库; 毒性质粒表达需要诱导,容易出现假阳性; 操作相对复杂	SpCas9 VQR	[34]
			SpCas9 EQR	[34]
			SpCas9 VRER	[34]
噬菌体辅助连续进化	可以同时进行突变和筛选	筛选过程只利用了Cas蛋白的结合能力, 所以筛选出的Cas蛋白更适合作为一种无切割活性的dCas蛋白	xCas9 3.7	[37]
晶体结构为基础的定点突变	结合晶体结构信息进行直接突变; 无需制备大量突变体	需要获得晶体结构; 突变位点选择不易	FnCas9 RHA	[62]
			SaCas9 KKH	[29]
			SpCas9-NG	[38]

方法	切割环境	筛选方向	特点	Cas蛋白	PAM	文献
生物信息学分析	无	正向	原始信息来自于基因组中spacer,结果可靠; 需要已知的spacer; 无法确保spacer来源的正确性; 获得的PAM信息可能不全面	SpCas9	NGG	[42]
				St1Cas9	NNAGAAW	[40]
				St3Cas9	NGGNG	[40]
				NmCas9	NNNNGATT	[60]
体内切割反应 (PAM 质粒库)	细菌	反向	该方法的PAM覆盖度高; 需要制备深度覆盖的质粒库; 需要构建Cas蛋白稳定表达的宿主; PAM逃逸受靶序列突变或DNA修复影响	SpCas9	NGG	[34,63]
				SpCas9 VQR	NGA	[34]
				SpCas9 EQR	NGAG	[34]
				SpCas9 VRER	NGCG	[34]
				SaCas9	NNGRRT	[34]
				SaCas9 KKH	NNNRRT	[29]
				CasX	TTCN	[31]
				CasY.1	TA	[31]
				St1Cas9	NNRGRA	[34,64]
				NmCas9	NNNNGNNT	[64]
体内切割反应 (crRNA 质粒库)	细菌	正向	提供了一种正向的筛选方式; 噬菌体基因组中展现出来的PAM有限; 构建向导RNA库的费用高于PAM库	FnCpf1	TTN	[25]
体外切割反应	体外	正向或反向	被切割模板量大,可以覆盖更多的潜在 PAM,快速; 需要纯化蛋白、靶向模板容易降解、体外条件可能导致PAM结果产生差异	SpCas9	NGG	[25,52,53]
				SpCas9-NG	NG	[38]
				SaCas9	NNGRRT	[25]
				FnCas9	NGG	[62]
				FnCas9 RHA	YG	[62]
				CjCas9	NNNVRYM	[65]
				St1Cas9	NNRRRA	[25]
					NNAGAAW	[53]
				St3Cas9	NGGNG	[60]
PAM-SCANR	细菌	正向	是一种正向筛选; 采用dCas蛋白,结果可能与以切割为基础分析获得的PAM有差异; 筛选可能会受到阻遏效应波动影响; 操作相对复杂	SpCas9	NGG	[54]
PAM-SCANR	细菌	正向		St1Cas9	NNAGAA	[54]
PAM-DOSE	人类细胞	正向	能够直接在人类细胞中进行Cas蛋白 PAM的确定; 采用正向筛选方式,不用制作大容量的 PAM库; 筛选方法依赖NHEJ修复	SpCas9	NGG	[55]
				SpCas9-NG	NG	[55]
				FnCpf1	YYN	[55]
				LbCpf1	YYN	[55]
				AsCpf1	YYN	[55]
				MbCpf1	YYN	[55]

CRISPR-Cas基因编辑系统升级：聚焦Cas蛋白和PAM

Next-generation CRISPR-Cas for genome editing: focusing on the Cas protein and PAM

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