CRISPR-Cas系统编辑丝状真菌的进展与挑战

肖晗; 刘宜欣

doi:10.12211/2096-8280.2020-078

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CRISPR-Cas系统编辑丝状真菌的进展与挑战

特约评述 | 更新时间：2025-03-20

- CRISPR-Cas系统编辑丝状真菌的进展与挑战
- Progress and challenge of the CRISPR-Cas system in gene editing for filamentous fungi
- 合成生物学 2021年2卷第2期页码：274-286
- 作者机构：
  
  上海交通大学生命科学技术学院，微生物代谢国家重点实验室，教育部代谢与发育科学国际合作联合实验室，上海 200240
- 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" ]
- 基金信息：
  
  国家重点研发项目(2018YFA0900600);国家自然科学基金(319713144;31600071);上海市自然科学基金(17ZR1448900;18ZR1420300)
- DOI：10.12211/2096-8280.2020-078
  中图分类号： Q81
- 收稿：2020-10-06，
  
  修回：2020-12-22，
  
  纸质出版：2021-04-30
- 稿件说明：
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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
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 DOI： 10.12211/2096-8280.2020-078.

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 DOI： 10.12211/2096-8280.2020-078.

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。

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 filam

entous 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.

关键词

Keywords

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