CRISPR-Cas系统编辑丝状真菌的进展与挑战

doi:10.12211/2096-8280.2020-078

摘要/Abstract

摘要：

丝状真菌是一类在蛋白分泌、活性次级代谢物生产、环境污染治理等方面起着重要作用的微生物，关于它们的各项基础和应用研究均高度依赖基因编辑平台。然而，丝状真菌的顶端生长、异核性、同源重组效率低和遗传标记匮乏等生理特点为构建这类微生物成熟的基因编辑平台带来挑战。近年来，基于RNA介导的CRISPR-Cas （clustered regularly interspaced short palindromic repeat/CRISPR-associated protein）系统在丝状真菌中得到越来越广泛的应用。由于构成简单、靶向特异，CRISPR-Cas系统极大促进了丝状真菌的基因编辑，包括基因插入、缺失、碱基转换和转录激活等。编辑的基因包括标记基因、非筛选标记的其他功能基因、功能未知的基因，甚至多个基因。编辑的尺度包括从1个碱基变化到缺失48 kb的基因簇。此外，借助精妙的同源重组供体设计和中断宿主NHEJ的关键基因，CRISPR-Cas系统能在特定位点引入精准修饰。本文围绕CRISPR-Cas系统的递送、体内表达、同源臂设计和宿主改造几方面重点介绍了该系统编辑丝状真菌近三年的进展。转化效率低和编辑效率低是现阶段CRISPR-Cas系统编辑丝状真菌存在的问题。针对这些问题，本文还讨论了可能的解决办法，为构建丝状真菌成熟的基因编辑平台提供了思路。

关键词: 丝状真菌, CRISPR-Cas, 同源重组, 基因编辑, 递送, 表达

Abstract:

Filamentous fungi are a group of microorganisms that play important roles in producing proteins (enzymes) and secondary metabolites as well as treating environmental pollutants. The basic and applied research on filamentous fungi, including identification of gene function and activation of silent gene cluster, relies heavily on gene editing. However, the apical growth, heterokaryosis, low efficiency of homologous recombination, and the lack of selective marker pose challenges for establishing gene editing platforms in filamentous fungi. In recent years, the RNA-mediated Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein (Cas) system has been widely employed in engineering filamentous fungi. Due to its simplicity and target specificity, the CRISPR-Cas system has assisted gene insertion, gene deletion, base conversion and transcriptional activation in this species. The edited targets can be single gene encoding a marker or enzyme with known or unknown function, and multiple genes as well, and the editing scale varies from one base to 48 kb. Furthermore, the CRISPR-Cas system allows precise modification at target site by introducing the cleverly designed homologous recombination donor and disrupting key genes in the non-homologous end joining (NHEJ). In this review, we comment research progress of the CRISPR-Cas system in gene editing for filamentous fungi that has been achieved in the past three years, with main focus on the delivery of CRISPR-Cas, in vivo expression of Cas protein and guide RNA (gRNA), the design of homologous recombination arms, and host modifications. The low efficiencies in both gene transformation and editing are still main challenges for CRISPR-Cas assisted gene editing in filamentous fungi, which is expected to be addressed by breakthroughs in fundamentals such as interactions between genotype and phenotype to discover genetic determinants.

Key words: filamentous fungi, CRISPR-Cas, homologous recombination, gene editing, delivery, expression

中图分类号:

肖晗, 刘宜欣. CRISPR-Cas系统编辑丝状真菌的进展与挑战[J]. 合成生物学, 2021, 2(2): 274-286.

XIAO Han, LIU Yixin. Progress and challenge of the CRISPR-Cas system in gene editing for filamentous fungi[J]. Synthetic Biology Journal, 2021, 2(2): 274-286.

图/表 4

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菌株	CRISPR-Cas存在形式	递送方式	表达策略	同源重组供体	宿主改造	编辑方式和效率
稻瘟病菌（Magnaporthe oryzae）	RNP	利用磷酸钙矿化的纳米颗粒承载RNP与原生质体共培养	体外表达并组装成有功能的RNP	—	—	NHEJ，中断编码Sytalone dehydratase的sdh基因的效率为20%^[23]
水稻恶苗病菌（Fusarium fujikuroi）	质粒	PEG介导的原生质体转化	内源表达水稻恶苗病菌密码子优化的Cas9，并用RNA聚合酶II型启动子加上自我剪切核酶的方式、RNA聚合酶III型U6或5S rRNA启动子分别表达gRNA	—	—	NHEJ， RNA聚合酶II型启动子加上自我剪切核酶的方式表达gRNA时，不能获得Fusarium cyclin C1编码基因fcc1的基因编辑突变体；用RNA聚合酶III型U6和5S rRNA启动子表达gRNA时， fcc1中断效率分别是37.5%和79.2%^[38]
嗜热毁丝霉（Myceliophthora thermophila）	PCR产物	PEG介导的原生质体转化	内源表达密码子优化的Cas12a并用U6启动子驱动gRNA的表达	PCR产物；两端靠近CRISPR-Cas切口处分别有500~600 bp的同源臂，中间包含抗性筛选标记基因表达盒	—	HDR，编辑单基因的效率为90%；编辑多个基因时，单基因发生编辑的效率为13%～41%^[35]；
黑曲霉（Aspergillus niger）	质粒	PEG介导的原生质体转化	利用可自主复制并丢失的质粒装载CRISPR-Cas系统，RNA聚合酶II型启动子加核酶的形式表达gRNA	质粒；插入片段两侧有CRISPR-Cas在基因组上产生切口附近的同源臂，同源臂两侧有和CRIPSR-Cas在基因组上识别区域一致的spacer	pyrG基因中断菌株	每转1 μg DNA获得2个有正确整合形式的转化子^[25]
黑曲霉（Aspergillus niger）	质粒	—	融合表达大鼠的胞嘧啶脱氨酶rAPOBEC1、黑曲霉密码子优化的nCas9和尿嘧啶糖基化抑制酶，用米曲霉U6启动子驱动gRNA的表达	—	—	将靶基因中特定位置的胞嘧啶转变为胸腺嘧啶，效率47.36%～100%^[31]
黑曲霉（Aspergillus niger）	质粒	PEG介导的原生质体转化	利用黑曲霉密码子优化的Cas9和内源5S rRNA基因启动子驱动gRNA的表达	PCR产物；两端和CRISPR-Cas切口处分别有40 bp的同源臂	kusA缺陷型菌株	HDR，效率为33.3%～100%^[47]
黑曲霉（Aspergillus niger）	质粒	PEG介导的原生质体转化	利用可自主复制的质粒装载CRISPR-Cas系统，内源表达Cas9，并用tRNA基因启动子驱动gRNA的表达	60 bp的寡核苷酸链，突变位点和PAM的距离不超过15 bp	kusA缺陷型菌株	HDR，效率为80%^[56]
烟曲霉（Aspergillus fumigatus）	质粒和RNA	PEG介导的原生质体转化	内源表达人密码子优化的Cas9和体外转录gRNA	PCR产物；两端靠近CRISPR-Cas切口处分别有35 bp的同源臂	—	HDR，效率为95%～100%^[53]
里氏木霉（Trichoderma reesei）	质粒和RNA	PEG介导的原生质体转化	内源表达里氏木霉优化的Cas9和体外转录成熟的gRNA	质粒；两端和CRISPR-Cas切口处分别有200 bp的同源臂	以表达Cas9的菌株为出发菌株	HDR，编辑内源一个假定的甲基转移酶基因lae1的效率为93%^[14]
灵芝（Ganoderma lucidum）	质粒	PEG介导的原生质体转化	利用灵芝密码子优化的Cas9和内源U6启动子驱动gRNA的表达，在gRNA 3′端加入HDV	PCR产物；两端和CRISPR-Cas切口处分别有1 kb的同源臂，中间缺失包括PAM在内的8 bp并引入终止密码子TGA	—	HDR，编辑细胞色素P450编码基因cyp5150l8的效率为每转化3×10⁷个原生质体获得2个基因编辑菌株^[15]

菌株	CRISPR-Cas存在形式	递送方式	表达策略	同源重组供体	宿主改造	编辑方式和效率
稻瘟病菌（Magnaporthe oryzae）	RNP	利用磷酸钙矿化的纳米颗粒承载RNP与原生质体共培养	体外表达并组装成有功能的RNP	—	—	NHEJ，中断编码Sytalone dehydratase的sdh基因的效率为20%^[23]
水稻恶苗病菌（Fusarium fujikuroi）	质粒	PEG介导的原生质体转化	内源表达水稻恶苗病菌密码子优化的Cas9，并用RNA聚合酶II型启动子加上自我剪切核酶的方式、RNA聚合酶III型U6或5S rRNA启动子分别表达gRNA	—	—	NHEJ， RNA聚合酶II型启动子加上自我剪切核酶的方式表达gRNA时，不能获得Fusarium cyclin C1编码基因fcc1的基因编辑突变体；用RNA聚合酶III型U6和5S rRNA启动子表达gRNA时， fcc1中断效率分别是37.5%和79.2%^[38]
嗜热毁丝霉（Myceliophthora thermophila）	PCR产物	PEG介导的原生质体转化	内源表达密码子优化的Cas12a并用U6启动子驱动gRNA的表达	PCR产物；两端靠近CRISPR-Cas切口处分别有500~600 bp的同源臂，中间包含抗性筛选标记基因表达盒	—	HDR，编辑单基因的效率为90%；编辑多个基因时，单基因发生编辑的效率为13%～41%^[35]；
黑曲霉（Aspergillus niger）	质粒	PEG介导的原生质体转化	利用可自主复制并丢失的质粒装载CRISPR-Cas系统，RNA聚合酶II型启动子加核酶的形式表达gRNA	质粒；插入片段两侧有CRISPR-Cas在基因组上产生切口附近的同源臂，同源臂两侧有和CRIPSR-Cas在基因组上识别区域一致的spacer	pyrG基因中断菌株	每转1 μg DNA获得2个有正确整合形式的转化子^[25]
黑曲霉（Aspergillus niger）	质粒	—	融合表达大鼠的胞嘧啶脱氨酶rAPOBEC1、黑曲霉密码子优化的nCas9和尿嘧啶糖基化抑制酶，用米曲霉U6启动子驱动gRNA的表达	—	—	将靶基因中特定位置的胞嘧啶转变为胸腺嘧啶，效率47.36%～100%^[31]
黑曲霉（Aspergillus niger）	质粒	PEG介导的原生质体转化	利用黑曲霉密码子优化的Cas9和内源5S rRNA基因启动子驱动gRNA的表达	PCR产物；两端和CRISPR-Cas切口处分别有40 bp的同源臂	kusA缺陷型菌株	HDR，效率为33.3%～100%^[47]
黑曲霉（Aspergillus niger）	质粒	PEG介导的原生质体转化	利用可自主复制的质粒装载CRISPR-Cas系统，内源表达Cas9，并用tRNA基因启动子驱动gRNA的表达	60 bp的寡核苷酸链，突变位点和PAM的距离不超过15 bp	kusA缺陷型菌株	HDR，效率为80%^[56]
烟曲霉（Aspergillus fumigatus）	质粒和RNA	PEG介导的原生质体转化	内源表达人密码子优化的Cas9和体外转录gRNA	PCR产物；两端靠近CRISPR-Cas切口处分别有35 bp的同源臂	—	HDR，效率为95%～100%^[53]
里氏木霉（Trichoderma reesei）	质粒和RNA	PEG介导的原生质体转化	内源表达里氏木霉优化的Cas9和体外转录成熟的gRNA	质粒；两端和CRISPR-Cas切口处分别有200 bp的同源臂	以表达Cas9的菌株为出发菌株	HDR，编辑内源一个假定的甲基转移酶基因lae1的效率为93%^[14]
灵芝（Ganoderma lucidum）	质粒	PEG介导的原生质体转化	利用灵芝密码子优化的Cas9和内源U6启动子驱动gRNA的表达，在gRNA 3′端加入HDV	PCR产物；两端和CRISPR-Cas切口处分别有1 kb的同源臂，中间缺失包括PAM在内的8 bp并引入终止密码子TGA	—	HDR，编辑细胞色素P450编码基因cyp5150l8的效率为每转化3×10⁷个原生质体获得2个基因编辑菌株^[15]

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