Engineered DNase-inactive Cpf1 variants to improve targeting scope for base editing in E.coli

The development of base editing(BE)technology has opened a new avenue for research studies in bacteriology,particularly for bacterial species in which the DNA double-strand breaks(DSBs)introduced by CRISPR/Cas system would lead to cell death.However,a major limitation of BE-mediated gene editing is the restricted editable sites in the target bacterial genome due to highly diverse genomic compositions,such as GC content.Herein,we developed a broad-spectrum DNase-inactive Cpf1(dCpf1)variant from Francisella novicida(bsdFnCpf1)through directed evolution.The resulting optimized mutant showed a substantially expanded targeting range,including previously non-canonical protospacer-adjacent motifs(PAMs),especially the GC-rich PAMs.Cytidine deaminase APOBEC1 and uracil DNA glycosylase inhibitor(UGI)were fused with bsdFnCpf1 to achieve specific C to T mutations at multiple target sites with canonical or non-canonical PAMs in the E.coli genome without compromising cell growth.We anticipate that bsdFnCpf1 could be applied for multiplex gene regulation and BE in species that have been reported to be suitable for Cpf1.