CRISPR/Cas基因编辑技术及其在微藻研究中的应用

doi:10.13560/j.cnki.biotech.bull.1985.2021-0877

摘要/Abstract

摘要：

微藻由于在医药、食品、可再生燃料和化学原料等方面的潜力,受到了研究者越来越多的关注和青睐。然而,合适基因编辑方法和转化工具的缺乏,使得微藻基因工程的进展还相对比较缓慢。随着分子生物和基因编辑技术的发展,CRISPR 技术凭借简便、特异和高效的优势,逐渐成为探讨基因功能、提高植物育种和增加代谢物产物等研究的有力手段。基于此,本文综述了CRISPR/Cas 的2 种主要类型,重点论述了其在微藻领域中的应用进展,并总结了CRISPR 技术在微藻应用中所存在的问题,期望为以后的研究提供启发和参考。

关键词: 基因编辑, CRISPR/Cas, 微藻, Cas9, 转化

Abstract:

Due to their potentials in medicine,food,renewable fuels and chemical raw materials,microalgae has attracted more and more attentions from current researchers. However,the progress of microalgae in genetic engineering is relatively slow because it is lack of suitable gene editing methods and transformation tools. With the development of molecular biology and gene editing technology,CRISPR technology has emerged as a powerful method for studying gene function,improving plant breeding and increasing metabolite products with its advantages of simplicity,specificity and efficiency. Based on these,we introduced the two main types of CRISPR/Cas,focusing on the application progress of CRISPR in microalgae,and summarized the issues existing in the application of CRISPR technology in microalgae,aiming to provide inspiration and reference for future research.

Key words: gene editing, CRISPR/Cas, microalgae, Cas9, transformation

陈映丹, 张扬, 夏嫱, 孙虹霞. CRISPR/Cas基因编辑技术及其在微藻研究中的应用[J]. 生物技术通报, 2022, 38(5): 257-268.

CHEN Ying-dan, ZHANG Yang, XIA Qiang, SUN Hong-xia. Gene Editing Technology of CRISPR/Cas and Its Applications in Microalgae Research[J]. Biotechnology Bulletin, 2022, 38(5): 257-268.

图/表 3

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特征Characteristic	Cas9	Cpf1
CRISPR分类	Class 2,type II Class 2,type V
来源	Streptococcus pyogenes	Francisella novicida Acidaminococcussp. Lachnospiraceae bacterium Moraxella bovoculi
核酸酶结构域	HNH、RuvC	RuvC
CRISPR-RNA	crRNA、tracrRNA	crRNA
PAM	NGG	TTTN
切割特点	平末端	黏性末端
RNAase活性	无	有

特征Characteristic	Cas9	Cpf1
CRISPR分类	Class 2,type II Class 2,type V
来源	Streptococcus pyogenes	Francisella novicida Acidaminococcussp. Lachnospiraceae bacterium Moraxella bovoculi
核酸酶结构域	HNH、RuvC	RuvC
CRISPR-RNA	crRNA、tracrRNA	crRNA
PAM	NGG	TTTN
切割特点	平末端	黏性末端
RNAase活性	无	有

类别 Category	微藻类型 Types of microalgae	方法 Method	目标基因 Target gene	相关内容 Related information	参考文献 Reference
衣藻	Chlamydonomas reinhardtii CC503	CRISPR/Cas9	FKB12	CRISPR/Cas9系统首次应用在微藻	[40]
	Chlamydonomas reinhardtii CC-124	CRISPR/Cas9、RNP	MAA7、CpSRP43、ChlM	靶向敲除或敲入,并通过NHEJ修复	[41]
	Chlamydonomas reinhardtii CC-4349	CRISPR/Cas9、RNP	ZEP、CpFTSY	实现了双基因敲除,产生突变体	[42]
	Chlamydonomas reinhardtii CC-4349	CRISPR/Cas9、RNP	ZEP	ZEP敲除突变体能够产生大量的叶黄素和玉米黄质	[43]
	Chlamydonomas reinhardtii CC-2931 Chlamydonomas reinhardtii CC-1883	CRISPR / Cpf1、RNP	FKB12、CpFTSY、CpSRP43、PHT7	Cpf1核酸酶首次应用于莱茵衣藻	[44]
	Chlamydonomas reinhardtii CC-400	CRISPR/dCas9	PEPC1、RFP	CRISPRi系统首次应用在莱茵衣藻	[45]
	Chlamydonomas reinhardtii CC-125	CRISPR/Cas9、RNP	MAA7	优化CRISPR RNP流程,提高基因编辑效率	[46]
蓝藻	Synechococcus elongatus UTEX 2973	CRISPR/Cas9	nblA	这是在蓝藻中使用CRISPR / Cas9基因组编辑的第一份报告	[49]
	Synechococcus elongatus PCC 7942	CRISPR/Cas9	glgc	glgC缺失突变体产生更高水平的琥珀酸酯	[50]
	Synechococcus UTEX 2973	CRISPR/Cpf1	psbA1、nblA	比较Cpf1和Cas9的毒性;Cpf1是蓝藻基因组编辑的合适核酸酶	[51]
	Synechocystis 6803 Anabaena 7120	CRISPR/Cpf1	nblA、nifH	缺失突变、点突变以及插入突变,研究了Cpf1的多功能性	[51]
	Synechcocystis sp. PCC 6803	CRISPR/dCas9	phaE、glgC	抑制碳存储化合物聚羟基丁酸酯（PHB）和糖原的形成。	[52]
	Synechococcus elongatus PCC 7942	CRISPR/dCas9	glgc、sdhA、sdhB	增加了琥珀酸产量	[53]
	Anabaena sp. PCC 7120	CRISPR/dCas9	glnA	成功微调glnA的表达水平;控制铵的生产。	[54]
	Synechocystis sp. PCC 6803	CRISPR/dCas9	PlsX	抑制必需的酰基转移酶PlsX可将脂肪醇滴度提高3倍。	[57]
	Synechococcus elongatus PCC 7942	CRISPR/dCpf1	nblA、acnB、cpcB2	使用CRISPR-dCas12a改善光合角鲨烯的生产	[56]
硅藻	Phaeodactylum tricornutum	CRISPR/Cas9	CpSRP54	CRISPR/Cas9首次在硅藻的应用	[58]
	Thalassiosira pseudonana	CRISPR/Cas9	urease	脲酶基因的精确删除	[59]
	Phaeodactylum tricornutum	CRISPR/Cas9	vtc2、pho4	优化方法,产生单等位基因突变和双等位基因突变	[60]
	Thalassiosira pseudonana	CRISPR/Cas9	TpθCA3	使用Cas9切口酶介导的基因组编辑,获得了海洋硅藻突变体	[61]
	Phaeodactylum tricornutum	CRISPR/Cas9	NR、GS-2、cGOGAT	新的Cas9附加体设计减少了生产和筛选突变菌株的时间	[62]
其他	Nannochloropsis oceanica IMET1	CRISPR/Cas9	Nitrate reductase gene、HygR	含油微藻的基因编辑,突变菌株敲除效率提高	[63]
	Nannochloropsis gaditana	CRISPR/Cas9	ZnCys	微调ZnCys表达,优化脂质生产	[64]
	Chlorella vulgarisFSP-E	CRISPR/Cas9	fad3	CRISPR / Cas9系统是首次应用于小球藻FSP-E	[65]

类别 Category	微藻类型 Types of microalgae	方法 Method	目标基因 Target gene	相关内容 Related information	参考文献 Reference
衣藻	Chlamydonomas reinhardtii CC503	CRISPR/Cas9	FKB12	CRISPR/Cas9系统首次应用在微藻	[40]
	Chlamydonomas reinhardtii CC-124	CRISPR/Cas9、RNP	MAA7、CpSRP43、ChlM	靶向敲除或敲入,并通过NHEJ修复	[41]
	Chlamydonomas reinhardtii CC-4349	CRISPR/Cas9、RNP	ZEP、CpFTSY	实现了双基因敲除,产生突变体	[42]
	Chlamydonomas reinhardtii CC-4349	CRISPR/Cas9、RNP	ZEP	ZEP敲除突变体能够产生大量的叶黄素和玉米黄质	[43]
	Chlamydonomas reinhardtii CC-2931 Chlamydonomas reinhardtii CC-1883	CRISPR / Cpf1、RNP	FKB12、CpFTSY、CpSRP43、PHT7	Cpf1核酸酶首次应用于莱茵衣藻	[44]
	Chlamydonomas reinhardtii CC-400	CRISPR/dCas9	PEPC1、RFP	CRISPRi系统首次应用在莱茵衣藻	[45]
	Chlamydonomas reinhardtii CC-125	CRISPR/Cas9、RNP	MAA7	优化CRISPR RNP流程,提高基因编辑效率	[46]
蓝藻	Synechococcus elongatus UTEX 2973	CRISPR/Cas9	nblA	这是在蓝藻中使用CRISPR / Cas9基因组编辑的第一份报告	[49]
	Synechococcus elongatus PCC 7942	CRISPR/Cas9	glgc	glgC缺失突变体产生更高水平的琥珀酸酯	[50]
	Synechococcus UTEX 2973	CRISPR/Cpf1	psbA1、nblA	比较Cpf1和Cas9的毒性;Cpf1是蓝藻基因组编辑的合适核酸酶	[51]
	Synechocystis 6803 Anabaena 7120	CRISPR/Cpf1	nblA、nifH	缺失突变、点突变以及插入突变,研究了Cpf1的多功能性	[51]
	Synechcocystis sp. PCC 6803	CRISPR/dCas9	phaE、glgC	抑制碳存储化合物聚羟基丁酸酯（PHB）和糖原的形成。	[52]
	Synechococcus elongatus PCC 7942	CRISPR/dCas9	glgc、sdhA、sdhB	增加了琥珀酸产量	[53]
	Anabaena sp. PCC 7120	CRISPR/dCas9	glnA	成功微调glnA的表达水平;控制铵的生产。	[54]
	Synechocystis sp. PCC 6803	CRISPR/dCas9	PlsX	抑制必需的酰基转移酶PlsX可将脂肪醇滴度提高3倍。	[57]
	Synechococcus elongatus PCC 7942	CRISPR/dCpf1	nblA、acnB、cpcB2	使用CRISPR-dCas12a改善光合角鲨烯的生产	[56]
硅藻	Phaeodactylum tricornutum	CRISPR/Cas9	CpSRP54	CRISPR/Cas9首次在硅藻的应用	[58]
	Thalassiosira pseudonana	CRISPR/Cas9	urease	脲酶基因的精确删除	[59]
	Phaeodactylum tricornutum	CRISPR/Cas9	vtc2、pho4	优化方法,产生单等位基因突变和双等位基因突变	[60]
	Thalassiosira pseudonana	CRISPR/Cas9	TpθCA3	使用Cas9切口酶介导的基因组编辑,获得了海洋硅藻突变体	[61]
	Phaeodactylum tricornutum	CRISPR/Cas9	NR、GS-2、cGOGAT	新的Cas9附加体设计减少了生产和筛选突变菌株的时间	[62]
其他	Nannochloropsis oceanica IMET1	CRISPR/Cas9	Nitrate reductase gene、HygR	含油微藻的基因编辑,突变菌株敲除效率提高	[63]
	Nannochloropsis gaditana	CRISPR/Cas9	ZnCys	微调ZnCys表达,优化脂质生产	[64]
	Chlorella vulgarisFSP-E	CRISPR/Cas9	fad3	CRISPR / Cas9系统是首次应用于小球藻FSP-E	[65]