Systems Analysis of Highly Multiplexed CRISPR-Base Editing in Streptomycetes

Abstract

CRISPRtools, especially Cas9n-sgRNA guided cytidine deaminasebase editors such as CRISPR-BEST, have dramatically simplified geneticmanipulation of streptomycetes. One major advantage of CRISPR baseediting technology is the possibility to multiplex experiments ingenomically instable species. Here, we demonstrate scaled up Csy4based multiplexed genome editing using CRISPR-mcBEST in Streptomyces coelicolor. We evaluated the systemby simultaneously targeting 9, 18, and finally all 28 predicted specializedmetabolite biosynthetic gene clusters in a single experiment. We presentimportant insights into the performance of Csy4 based multiplexedgenome editing at different scales. Using multiomics analysis, weinvestigated the systems wide effects of such extensive editing experimentsand revealed great potentials and important bottlenecks of CRISPR-mcBEST.The presented analysis provides crucial data and insights toward thedevelopment of multiplexed base editing as a novel paradigm for highthroughput engineering of Streptomyces chassis andbeyond.