Synthetic Biology Journal ›› 2021, Vol. 2 ›› Issue (2): 274-286.DOI: 10.12211/2096-8280.2020-078
• Invited Review • Previous Articles Next Articles
Han XIAO, Yixin LIU
Received:
2020-10-06
Revised:
2020-12-22
Online:
2021-04-30
Published:
2021-04-29
Contact:
Han XIAO
肖晗, 刘宜欣
通讯作者:
肖晗
作者简介:
肖晗(1985—),女,博士,副研究员,主要从事合成生物学、基因编辑和代谢工程研究。E-mail:smallhan@sjtu.edu.cn
基金资助:
CLC Number:
Han XIAO, Yixin LIU. Progress and challenge of the CRISPR-Cas system in gene editing for filamentous fungi[J]. Synthetic Biology Journal, 2021, 2(2): 274-286.
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.
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Fig. 1 CRISPR-Cas system assisted gene editing for filamentous fungi(RNP—ribonucleoprotein; BM-RNP—biomimetic mineralized RNP; PEG—polyethylene glycerol; AMT—agrobacterium-mediated transformation; DSB—double stranded break; NHEJ—non-homologous end joining; HR—homologous recombination; APOBEC1—apolipoprotein B mRNA editing enzyme; VPR—VP64-p65-Rta, a tripartite transcriptional activator domain; CRISPRa—CRISPR activation)
Fig. 2 Strategies for in vivo expression of the CRISPR-Cas system in filamentous fungi(For in vivo expression of Cas protein, strategies include codon-optimization, adopting constitutive promoter for driving the expression of Cas protein, and incorporating intron sequence at the 5' end of cas gene. For in vivo expression of gRNA, strategies include adopting polymerase II promoter together with the self-cleaving ribozymes for gRNA expression, adopting polymerase III promoter for gRNA expression, and adopting polymerase III promoter together with the self-cleaving ribozyme for gRNA expression)
Fig. 3 Design of homologous recombination donor(For linearized DNA, it is flanked by 39 bp HR arms, which are within 5 kb from DSB as generated by CRIPSR-Cas. For circular DNA, it is flanked by HR arms with the size from 0.5 to 2 kb, and a selective marker is usually contained in the middle. For oligo, the intended mutation and PAM sequence are interspaced by no more than 7 bp)
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%[ |
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%[ |
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%[ |
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[ |
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%[ |
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%[ |
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%[ |
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%[ |
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%[ |
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[ |
Tab. 1 Examples of CRISPR-Cas system assisted gene editing in filamentous fungi.
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%[ |
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%[ |
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%[ |
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[ |
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%[ |
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%[ |
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%[ |
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%[ |
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%[ |
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[ |
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