| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | A Streptomyces-codon-optimized cas9 gene was synthesized in our previous study6, DNAs encoding the remaining components (APOBEC1-Cas9_N_terminal-UGI for CBE; TadA-TadA*-Cas9_N_terminal for ABE) to complete the base editors were codon-optimized for Streptomyces, then together with the rpsLp and fdt the designed sequences were synthesized by GenScript and provided on the vector pUC57 to create transitional plasmids pUC57-SCBE-wocas9 and pUC57-SABE-wocas9, respectively. DNAs encoding the remaining portion of the nCas9 or dCas9 were PCR amplified from pWHU26506 and inserted into the above transitional plasmids by Gibson assembly. The common D10A mutation of nCas9 and dCas9 was incorporated in the synthesized Cas9 N-terminal; the H840A mutation of dCas9 was introduced by an additional overlap PCR. | Get A Quote |
Conventional CRISPR/Cas genetic manipulation has been profitably applied to the genus Streptomyces, the most prolific bacterial producers of antibiotics. However, its reliance on DNA double-strand break (DSB) formation leads to unacceptably low yields of desired recombinants. We have adapted for Streptomyces recently-introduced cytidine base editors (CBEs) and adenine base editors (ABEs) which enable targeted C-to-T or A-to-G nucleotide substitutions, respectively, bypassing DSB and the need for a repair template. We report successful genome editing in Streptomyces at frequencies of around 50% using defective Cas9-guided base editors and up to 100% by using nicked Cas9-guided base editors. Furthermore, we ... More
