Gene editing in bacteria via SpCas9/sgRNA ribonucleoprotein complexes

Abstract

Gene editing has been revolutionized by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas technology in a variety of organisms. In bacteria, however, CRISPR-Cas still holds many caveats such as high toxicity of Cas9 and off-target editing effects. In this work we develop a system for the incorporation of Cas9/single guide RNA ribonucleoprotein complexes in bacteria and their successful application for gene editing via homologous recombination. Targeting of a green fluorescent protein (GFP) reporter allows for easy verification of gene-edition via conversion to blue-fluorescent protein (BFP), mediated by the well characterized 196T > C (Tyr66His) mutation.