CRISPR/Cas12a toolbox for genomic manipulation inMethanosarcina acetivorans

Abstract

AbstractMethanogenic archaea play an important role in the global carbon cycle and are regarded as promising host organisms for the biotechnological generation of fuels and chemicals from one-carbon substrates.Methanosarcina acetivoransis extensively studied as a model methanogen due to the availability of genetic tools and its versatile substrate range. Although genome editing inM. acetivoransvia CRISPR/Cas9 has already been demonstrated, we now describe a user-friendly CRISPR/Cas12a toolbox that recognizes a T-rich (5′-TTTV) PAM sequence. This new system can manage deletions of 3500 bp (i.e., knockout of the entirefrhADGBoperon) and heterologous gene insertions with 80% efficiency observed in ten PurRtransformants. Our CRISPR/Cas12a system also enables multiplex genome editing at high efficiency, which helps speed up genetic engineering. Deletions of 100 bp generated on two separate sites of the genome yielded 8/8 correctly edited transformants. Simultaneous gene deletion (100 bp) and replacement (100-bp region replaced by the 2400-bpuidAexpression cassette) at a separate site was achieved, with 3/6 of transformants being edited correctly. In combination with the Cas9-based system, our CRISPR/Cas12a toolbox enables targeted genome editing at two sites (guanine-rich and thymine-rich, respectively) and, in so doing, hastens the overall genetic engineering of theMethanosarcinalesspecies.