Dalia Triana N, Yoon Soo Hun, Galli Elisa, Barre Francois-Xavier, Waters Christopher M, Dalia Ankur B
Department of Biology, Indiana University, Bloomington, IN 47401, USA.
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.
Nucleic Acids Res. 2017 Jul 7;45(12):7527-7537. doi: 10.1093/nar/gkx496.
Recently, we described a method for multiplex genome editing by natural transformation (MuGENT). Mutant constructs for MuGENT require large arms of homology (>2000 bp) surrounding each genome edit, which necessitates laborious in vitro DNA splicing. In Vibrio cholerae, we uncover that this requirement is due to cytoplasmic ssDNA exonucleases, which inhibit natural transformation. In ssDNA exonuclease mutants, one arm of homology can be reduced to as little as 40 bp while still promoting integration of genome edits at rates of ∼50% without selection in cis. Consequently, editing constructs are generated in a single polymerase chain reaction where one homology arm is oligonucleotide encoded. To further enhance editing efficiencies, we also developed a strain for transient inactivation of the mismatch repair system. As a proof-of-concept, we used these advances to rapidly mutate 10 high-affinity binding sites for the nucleoid occlusion protein SlmA and generated a duodecuple mutant of 12 diguanylate cyclases in V. cholerae. Whole genome sequencing revealed little to no off-target mutations in these strains. Finally, we show that ssDNA exonucleases inhibit natural transformation in Acinetobacter baylyi. Thus, rational removal of ssDNA exonucleases may be broadly applicable for enhancing the efficacy and ease of MuGENT in diverse naturally transformable species.
最近，我们描述了一种通过自然转化进行多重基因组编辑的方法（MuGENT）。MuGENT的突变构建体需要在每个基因组编辑周围有大的同源臂（>2000 bp），这使得体外DNA剪接工作繁重。在霍乱弧菌中，我们发现这种要求是由于细胞质单链DNA外切核酸酶，它会抑制自然转化。在单链DNA外切核酸酶突变体中，一个同源臂可以减少到低至40 bp，同时在没有顺式选择的情况下仍以约50%的速率促进基因组编辑的整合。因此，编辑构建体在单个聚合酶链反应中产生，其中一个同源臂是寡核苷酸编码的。为了进一步提高编辑效率，我们还开发了一种用于错配修复系统瞬时失活的菌株。作为概念验证，我们利用这些进展快速突变了类核结合蛋白SlmA的10个高亲和力结合位点，并在霍乱弧菌中产生了12个双鸟苷酸环化酶的十二重突变体。全基因组测序显示这些菌株中几乎没有脱靶突变。最后，我们表明单链DNA外切核酸酶抑制拜氏不动杆菌的自然转化。因此，合理去除单链DNA外切核酸酶可能广泛适用于提高MuGENT在各种自然可转化物种中的功效和简便性。
