构建CRISPR-Cas9介导的耻垢分枝杆菌基因组高效删除系统

CRISPR-Cas9-assisting efficient and sequential genome deletions in Mycobacterium smegmatis

摘要:

【背景】耻垢分枝杆菌具有生长迅速和非致病性的特点，可作为结核分枝杆菌致病机理研究替代菌株和类固醇激素生产的工程菌，但目前耻垢分枝杆菌中缺乏高效率的基因组敲除方法。【目的】基于CRISPR-Cas9介导的定点、高效的DNA切割能力，构建耻垢分枝杆菌染色体DNA片段无痕敲除系统。【方法】构建了包含四环素诱导型启动子驱动的密码子优化的cas9基础载体pCas9101，在双侧同源臂长度约为1 kb条件下选用合适的gRNA表达模块，分别测试了对耻垢分枝杆菌mc2155染色体上的3β-羟基类固醇脱氢酶基因(MSMEG_5228，1 071 bp)和胆固醇降解基因簇(MSMEG_5990-MSMEG_6043，约48 kb)敲除效率，使用相同大小的同源臂以经典p2NIL-pGOAL方法进行对照，并计算效率。【结果】使用CRISPR-Cas9方法对耻垢分枝杆菌mc2155的3β-羟基类固醇脱氢酶基因敲除效率为22%，胆固醇降解基因簇敲除效率也达到18%，两者连续敲除效率为4%。但对照p2NIL-pGOAL方法未能获得目标DNA片段敲除的菌株。【结论】本文建立的基于CRISPR-Cas9的耻垢分枝杆菌基因组无痕敲除系统显示出较高的敲除效率，该方法可为耻垢分枝杆菌后续研究提供快速高效的基因组操作方法。

Abstract:

[Background] For its fast growing and non-pathogenic property, Mycobacterium smegmatis is used as a model strain for pathogenic Mycobacterium tuberculosis and a workhorse to produce steroid hormones. But it lacks efficient genome deletion system. [Objective] In this study, we established a high efficiency genome deletion system in M. smegmatis assisted by CRISPR-Cas9. [Methods] The Cas9 backbone plasmid pCas9101 was constructed with a tetracycline-inducible codon-optimized cas9 expression operon. Approximate 1 kb flanked homologous arms and proper gRNA operon were used to construct vectors to study the deletion efficiency on 3β-hydroxysteroid dehydrogenase (MSMEG_5228, 1 071 bp) encoding gene and cholesterol degradation gene cluster (MSMEG_5990-MSMEG_6043, about 48 kb) in M. smegmatis mc2155. The classical p2NIL-pGOAL method with same homologous arms was used as the control to delete the two same DNA segments in M. smegmatis mc2155. Their deletion efficiency were calculated and compared. [Results] When using CRISPR-Cas9 assisted method, 22% and 18% clones showed the expected deletions in MSMEG_5228 gene and cholesterol-degrading gene cluster, respectively. Even the sequential deletion efficiency on both segments can reach 4%. However, no expected deletion mutants were obtained in our experiments with p2NIL-pGOAL method. [Conclusion] This CRISPR-Cas9 assisted system can facilitate genome deletion in M. smegmatis mc2155, and provide a fast and efficient genome manipulation approach for Mycobacterium in the future.