CRISPR-Cas系统编辑丝状真菌的进展与挑战

肖晗, 刘宜欣

合成生物学 ›› 2021, Vol. 2 ›› Issue (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。

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