Multiplex Single-Nucleotide Microbial Genome Editing Achieved by CRISPR-Cas9 Using 5 & PRIME;-End-Truncated sgRNAs

Abstract

Multiplex genome editing with CRISPR-Cas9 offers a cost-effectivesolution for time and labor savings. However, achieving high accuracyremains a challenge. In an Escherichia coli modelsystem, we achieved highly efficient single-nucleotide level simultaneousediting of the galK and xylB genesusing the 5 & PRIME;-end-truncated single-molecular guide RNA (sgRNA)method. Furthermore, we successfully demonstrated the simultaneousediting of three genes (galK, xylB, and srlD) at single-nucleotide resolution. Toshowcase practical application, we targeted the cI ( 857 ) and ilvG genes inthe genome of E. coli. While untruncated sgRNAs failedto produce any edited cells, the use of truncated sgRNAs allowed usto achieve simultaneous and accurate editing of these two genes withan efficiency of 30%. This enabled the edited cells to retain theirlysogenic state at 42 & DEG;C and effectively alleviated l-valine toxicity. These results suggest that our truncated sgRNAmethod holds significant potential for widespread and practical usein synthetic biology.