Harnessing CRISPR-Cas9 for genome editing in Streptococcus pneumoniae D39V.

Harnessing CRISPR-Cas9 for genome editing in Streptococcus pneumoniae D39V. Appl Environ Microbiol. 2021 Jan 04;: Authors: Synefiaridou D, Veening JW Abstract CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by detection and cleavage of invading foreign DNA. Modified versions of this system can be exploited as a biotechnological tool for precise genome editing at a targeted locus. Here, we developed a replicative plasmid that carries the CRISPR-Cas9 system for RNA-programmable, genome editing by counterselection in the opportunistic human pathogen Streptococcus pneumoniae. Specifically, we demonstrate an approach for making targeted, marker-less gene knockouts and large genome deletions. After a precise double-stranded break (DSB) is introduced, the cells' DNA repair mechanism of homology-directed repair (HDR) pathway is being exploited to select successful transformants. This is achieved through the transformation of a template DNA fragment that will recombine in the genome and eliminate recognition of the target of the Cas9 endonuclease. Next, the newly engineered strain can be easily cured from the plasmid that is temperature-sensitive for replication, by growing it at the non-permissive temperature. This allows for consecutive rounds of genome editing. Using this system, we engineered a strain with three major virulence factors deleted. The here developed approaches could be pote...
Source: Applied and Environmental Microbiology - Category: Microbiology Authors: Tags: Appl Environ Microbiol Source Type: research