CRISPR-dCas9 mediated cytosine deaminase base editing in bacillus subtilis

Sili Yu, Marcus A. Price, Yu Wang, Yang Liu, Yanmei Guo, Xiaomeng Ni, Susan J. Rosser, Changhao Bi, Meng Wang

Research output: Contribution to journalArticlepeer-review

Abstract

Base editing technology based on clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) is a recent addition to the family of CRISPR technologies. Compared with the traditional CRISPR/Cas9 technology, it does not rely on DNA double strand break and homologous recombination, and can realize gene inactivation and point mutation more quickly and simply. Herein, we first developed a base editing method for genome editing in Bacillus subtilis utilizing CRISPR/dCas9 (a fully nuclease-deficient mutant of Cas9 from S. pyogenes) and activation-induced cytidine deaminase (AID). This method achieved three and four loci simultaneous editing with editing efficiency up to 100% and 50%, respectively. Our base editing system in B. subtilis has a 5 nt editing window, which is similar to previously reported base editing in other microorganisms. We demonstrated that the plasmid curing rate is almost 100%, which is advantageous for multiple rounds of genome engineering in B. subtilis. Finally, we applied multiplex genome editing to generate a B. subtilis 168 mutant strain with eight inactive extracellular protease genes in just two rounds of base editing and plasmid curing, suggesting that it is a powerful tool for gene manipulation in B. subtilis and industrial applications in the future.
Original languageEnglish
Pages (from-to)1781-1789
JournalACS Synthetic Biology
Volume9
Issue number7
DOIs
Publication statusPublished - 17 Jun 2020

Keywords

  • CRISPR/dCas9
  • cytidine deaminase
  • genome editing
  • bacillus subtilis

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