Metabolic engineering of Escherichia coli BL21 strain using simplified CRISPR-Cas9 and asymmetric homology arms recombineering.

ABSTRACT:

Background

The recent CRISPR-Cas coupled with λ recombinase mediated genome recombineering has become a common laboratory practice to modify bacterial genomes. It requires supplying a template DNA with homology arms for precise genome editing. However, generation of homology arms is a time-consuming, costly and inefficient process that is often overlooked.

Results

In this study, we first optimized a CRISPR-Cas genome engineering protocol in the Escherichia coli (E. coli) BL21 strain and successfully deleted 10 kb of DNA from the genome in one round of editing. To further simplify the protocol, asymmetric homology arms were produced by PCR in a single step with two primers and then purified using a desalting column. Unlike conventional homology arms that are prepared through

SUBMITTER: Shukal S 

PROVIDER: S-EPMC8817497 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature