Synthesizing and deploying an improved CRISPR Cas12a knockout system for Synechococcus elongatus sp. PCC 7942

Abstract

CRISPR mediated genome editing may be one of the most profound biochemical breakthroughs of this century. As more research laboratories begin to incorporate CRISPR systems into their repertoire, having a CRISPR system that is easy to use becomes more important. In this study, we developed and demonstrated the efficacy of a CRISPR Cas12a system containing GFP whose implementation is more facile than its predecessors. This system was created primarily through restriction digestions and Gibson Assembly and was implemented via E. coli transformations and tri-parental mating. Using Synechococcus elongatus sp. PCC 7942 as our research strain, we demonstrated the efficacy of this system by deleting ndhF4, a part of the cyanobacterial CO₂ concentrating mechanism. In addition to developing a powerful tool for other genome editing, our ΔNdhF4 strain can be used as a genetic background for structure/function studies of NdhF4.