Login / Signup

An Efficient CRISPR/Cas12e System for Genome Editing in Sinorhizobium meliloti .

Guangqing LiuHuiying WangBaisong TongYali CuiSibylle C VoneschHuina DongDawei Zhang
Published in: ACS synthetic biology (2023)
CRISPR/Cas systems have been widely used in the precise and traceless genetic engineering of bacteria. Sinorhizobium meliloti 320 (SM320) is a Gram-negative bacterium with a low efficiency of homologous recombination but a strong ability to produce vitamin B 12 . Here, a CRISPR/Cas12e-based genome engineering toolkit, CRISPR/Cas12eGET, was constructed in SM320. The expression level of CRISPR/Cas12e was tuned through promoter optimization and the use of a low copy plasmid to adjust Cas12e cutting activity to the low homologous recombination efficiency of SM320, resulting in improved transformation and precision editing efficiencies. Furthermore, the accuracy of CRISPR/Cas12eGET was improved by deleting the ku gene involved in NHEJ repair in SM320. This advance will be useful for metabolic engineering and basic research on SM320, and it further provides a platform to develop the CRISPR/Cas system in strains where the efficiency of homologous recombination is low.
Keyphrases
  • crispr cas
  • genome editing
  • dna repair
  • dna damage
  • gram negative
  • multidrug resistant
  • genome wide
  • escherichia coli
  • gene expression
  • oxidative stress
  • copy number
  • wastewater treatment
  • long non coding rna