Multiplex genome editing using a dCas9-cytidine deaminase fusion in Streptomyces
Multiplex genome editing using a dCas9-cytidine deaminase fusion in Streptomyces
复制标题
在链霉菌中使用 dCas9-胞苷脱氨酶融合进行多重基因组编辑
DOI：
10.1007/s11427-019-1559-y
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发表时间：
2019-12-20
影响因子：
9.1
通讯作者：
Lu, Yinhua
中科院分区：
文献类型：
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作者：
Zhao, Yawei;Tian, Jinzhong;Lu, Yinhua
CRISPR/Cas介导的基因组编辑极大地促进了链霉菌基因功能的研究。然而，它不能有效地利用链霉菌与低同源重组（HR）能力。在此，在链霉菌中开发了脱氨酶辅助的碱基编辑器dCas 9-CDA-ULstr，其包含核酸酶缺陷型Cas9（dCas 9）、来自海岩巨虫的胞苷脱氨酶（PmCDAl）、尿嘧啶DNA糖基化酶抑制剂（UGI）和蛋白质降解标签（LVA标签）。使用dCas 9-CDA-ULstr，我们在天蓝色链霉菌中的靶位点处实现了单点、双点和三点突变（胞嘧啶至胸腺嘧啶取代），效率分别高达100%、60%和20%。这种碱基编辑器也被证明在生产免疫抑制剂雷帕霉素的工业菌株雷帕霉素链霉菌（Streptomyces rapamycinicus）中的碱基编辑是高效的。与来自胞苷脱氨酶rAPOBEC 1的碱基编辑器相比，PmCDA 1辅助的碱基编辑器dCas 9-CDA-ULstr可以高效率地编辑鸟苷之前的胞嘧啶，这对于编辑链霉菌基因组（具有高GC含量）是一个很大的优势。总的来说，碱基编辑器dCas 9-CDA-ULstr可用于链霉菌中的有效多重基因组编辑。由于基于dCas 9-CDA-ULstr的基因组编辑不依赖于HR介导的DNA修复，我们相信该技术将极大地促进具有弱HR能力的链霉菌菌株的功能基因组研究和代谢工程。
CRISPR/Cas-mediated genome editing has greatly facilitated the study of gene function in Streptomyces. However, it could not be efficiently employed in streptomycetes with low homologous recombination (HR) ability. Here, a deaminase-assisted base editor dCas9-CDA-ULstr was developed in Streptomyces, which comprises the nuclease-deficient Cas9 (dCas9), the cytidine deaminase from Petromyzon marinus (PmCDA1), the uracil DNA glycosylase inhibitor (UGI) and the protein degradation tag (LVA tag). Using dCas9-CDA-ULstr, we achieved single-, double- and triple-point mutations (cytosine-to-thymine substitutions) at target sites in Streptomyces coelicolor with efficiency up to 100%, 60% and 20%, respectively. This base editor was also demonstrated to be highly efficient for base editing in the industrial strain, Streptomyces rapamycinicus, which produces the immunosuppressive agent rapamycin. Compared with base editors derived from the cytidine deaminase rAPOBEC1, the PmCDA1-assisted base editor dCas9-CDA-ULstr could edit cytosines preceded by guanosines with high efficiency, which is a great advantage for editing Streptomyces genomes (with high GC content). Collectively, the base editor dCas9-CDA-ULstr could be employed for efficient multiplex genome editing in Streptomyces. Since the dCas9-CDA-ULstr-based genome editing is independent of HR-mediated DNA repair, we believe this technology will greatly facilitate functional genome research and metabolic engineering in Streptomyces strains with weak HR ability.