合成生物学 ›› 2023, Vol. 4 ›› Issue (4): 738-755.DOI: 10.12211/2096-8280.2022-076
林继聪1, 邹根2, 刘宏民1, 魏勇军1
收稿日期:
2022-12-28
修回日期:
2023-02-27
出版日期:
2023-08-31
发布日期:
2023-09-14
通讯作者:
刘宏民,魏勇军
作者简介:
基金资助:
Jicong LIN1, Gen ZOU2, Hongmin LIU1, Yongjun WEI1
Received:
2022-12-28
Revised:
2023-02-27
Online:
2023-08-31
Published:
2023-09-14
Contact:
Hongmin LIU, Yongjun WEI
摘要:
丝状真菌能够合成抗生素、色素、酶制剂、激素等多种天然产物,广泛应用于医药、化工、农业和基础生物学研究等领域。丝状真菌遗传背景复杂,阻碍了对其的进一步开发利用。基因组编辑是基于核酸酶对基因组位点特异性序列进行靶向切割,产生双链断裂,从而通过非同源末端连接或同源重组进行修复的技术。其中,CRISPR(clustered regularly interspaced short palindromic repeats)系统是目前使用最普遍的基因组编辑技术,已在丝状真菌遗传育种、基因改造和多种天然产物合成等方面进行了大量应用。本文总结了丝状真菌CRISPR/Cas的技术原理、元件表达、递送方式及该系统在次级代谢产物等研究中的应用。对于脱靶效应以及转化率低的问题,本文讨论了可能的解决方法。在此基础上,展望了基于CRISPR/Cas的基因组编辑技术在真菌基因功能表征、天然产物合成代谢途径解析与重构、高效丝状真菌底盘细胞构建、天然产物合成等方面的应用。
中图分类号:
林继聪, 邹根, 刘宏民, 魏勇军. CRISPR/Cas基因组编辑技术在丝状真菌次级代谢产物合成中的应用[J]. 合成生物学, 2023, 4(4): 738-755.
Jicong LIN, Gen ZOU, Hongmin LIU, Yongjun WEI. Application of CRISPR/Cas genome editing technology in the synthesis of secondary metabolites of filamentous fungi[J]. Synthetic Biology Journal, 2023, 4(4): 738-755.
丝状真菌 | sgRNA 启动子 | 表达方式 | 靶标基因 | 编辑效率 | 参考文献 |
---|---|---|---|---|---|
里氏木霉 | 体内表达密码子优化的Cas9和体外转录的sgRNA | ura5, lae1, vib1, clr2 | 单基因93%~100%;多基因4.2%~45% | [ | |
里氏木霉 | U6 | 体内表达密码子优化的Cas9和U6启动子驱动sgRNA1的表达 | ura5 | 8%~10% | [ |
里氏木霉 | 5S rRNA | 体内表达密码子优化的Cas9和5S rRNA驱动sgRNA的表达 | lae1 | 36.67% | [ |
里氏木霉 | 体外组装的核糖核蛋白(RNP)复合物 | ura5, lae1, cbh1, cbh2, eg1 | 56.52%~100% | [ | |
草酸青霉 | 5S rRNA | 体内表达密码子优化的Cas9和5S rRNA驱动sgRNA的表达 | bgl2, creA | 30%~80% | [ |
鲁本斯青霉 | U6, tRNA, utp25 | 体内表达密码子优化的Cas9与U6,tRNA和utp25启动子驱动sgRNA的表达;体外组装的核糖核蛋白(RNP)复合物 | pks17, roqA, lovF, hcpA, pcbA, penDE, chyA | 60%~100% | [ |
黑曲霉 | tRNA | 体内表达密码子优化的Cas9和tRNA驱动sgRNA的表达 | albA, olvA, glaA | 20%~93% | [ |
黑曲霉 | 5S rRNA | 体内表达密码子优化的Cas9和tRNA驱动sgRNA的表达 | albA, fum5, fum1 | 33%~100% | [ |
米曲霉 | U6 | 体内表达密码子优化的Cas9和U6启动子驱动sgRNA的表达 | wA, pyrG, yA | 10%~100% | [ |
米曲霉 | U6 | 携带Cas9和sgRNA的自主复制质粒 | wA, pyrG, yA | 55.6%~100% | [ |
烟曲霉 | 体内表达密码子优化的Cas9和体外转录的sgRNA | pksP, cnaA | 95%~100% | [ | |
藤仓镰刀菌 | PgpdA, U6, 5S rRNA | 体内表达密码子优化的Cas9与PgpdA 、U6、5S rRNA启动子驱动sgRNA的表达 | fcc1, ura3, ppt1 | 37.5%~79.2% | [ |
Glarea lozoyensis | 5S rRNA | 体内表达密码子优化的Cas9和5S rRNA驱动sgRNA的表达 | gloA, gloF | 28% | [ |
嗜热毁丝霉 | U6 | 体内表达密码子优化的Cas9和U6启动子驱动sgRNA的表达 | cre-1, res-1, gh1-1, alp-1 | 单基因95%; 多基因22%~70% | [ |
嗜热毁丝霉 | U6 | 体内表达密码子优化的Cas12a和U6启动子驱动sgRNA的表达 | cre-1, res-1, gh1-1 | 单基因90%; 多基因13%~41% | [ |
稻瘟病菌 | 体外组装的Cas12a核糖核蛋白(RNP)复合物 | BUF1, FKBP12, FTR1, BAS4, AVRPI9 | 25%~77% | [ |
表1 丝状真菌中CRISPR/Cas基因组编辑系统的应用
Table 1 Application of CRISPR/Cas genome editing system in filamentous fungi
丝状真菌 | sgRNA 启动子 | 表达方式 | 靶标基因 | 编辑效率 | 参考文献 |
---|---|---|---|---|---|
里氏木霉 | 体内表达密码子优化的Cas9和体外转录的sgRNA | ura5, lae1, vib1, clr2 | 单基因93%~100%;多基因4.2%~45% | [ | |
里氏木霉 | U6 | 体内表达密码子优化的Cas9和U6启动子驱动sgRNA1的表达 | ura5 | 8%~10% | [ |
里氏木霉 | 5S rRNA | 体内表达密码子优化的Cas9和5S rRNA驱动sgRNA的表达 | lae1 | 36.67% | [ |
里氏木霉 | 体外组装的核糖核蛋白(RNP)复合物 | ura5, lae1, cbh1, cbh2, eg1 | 56.52%~100% | [ | |
草酸青霉 | 5S rRNA | 体内表达密码子优化的Cas9和5S rRNA驱动sgRNA的表达 | bgl2, creA | 30%~80% | [ |
鲁本斯青霉 | U6, tRNA, utp25 | 体内表达密码子优化的Cas9与U6,tRNA和utp25启动子驱动sgRNA的表达;体外组装的核糖核蛋白(RNP)复合物 | pks17, roqA, lovF, hcpA, pcbA, penDE, chyA | 60%~100% | [ |
黑曲霉 | tRNA | 体内表达密码子优化的Cas9和tRNA驱动sgRNA的表达 | albA, olvA, glaA | 20%~93% | [ |
黑曲霉 | 5S rRNA | 体内表达密码子优化的Cas9和tRNA驱动sgRNA的表达 | albA, fum5, fum1 | 33%~100% | [ |
米曲霉 | U6 | 体内表达密码子优化的Cas9和U6启动子驱动sgRNA的表达 | wA, pyrG, yA | 10%~100% | [ |
米曲霉 | U6 | 携带Cas9和sgRNA的自主复制质粒 | wA, pyrG, yA | 55.6%~100% | [ |
烟曲霉 | 体内表达密码子优化的Cas9和体外转录的sgRNA | pksP, cnaA | 95%~100% | [ | |
藤仓镰刀菌 | PgpdA, U6, 5S rRNA | 体内表达密码子优化的Cas9与PgpdA 、U6、5S rRNA启动子驱动sgRNA的表达 | fcc1, ura3, ppt1 | 37.5%~79.2% | [ |
Glarea lozoyensis | 5S rRNA | 体内表达密码子优化的Cas9和5S rRNA驱动sgRNA的表达 | gloA, gloF | 28% | [ |
嗜热毁丝霉 | U6 | 体内表达密码子优化的Cas9和U6启动子驱动sgRNA的表达 | cre-1, res-1, gh1-1, alp-1 | 单基因95%; 多基因22%~70% | [ |
嗜热毁丝霉 | U6 | 体内表达密码子优化的Cas12a和U6启动子驱动sgRNA的表达 | cre-1, res-1, gh1-1 | 单基因90%; 多基因13%~41% | [ |
稻瘟病菌 | 体外组装的Cas12a核糖核蛋白(RNP)复合物 | BUF1, FKBP12, FTR1, BAS4, AVRPI9 | 25%~77% | [ |
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