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Background  Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes.  Zymomonas mobilis  is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for  Z. mobilis , efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in  Z. mobilis  are still lacking.    Results  Using Cas12a (Cpf1) from  Francisella novicida , a recombinant strain with inducible  cas12a  expression for genome editing was constructed in  Z. mobilis  ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR–Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR–Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR–Cas12a system was employed to introduce a heterologous lactate dehydrogenase into  Z. mobilis  with a recombinant lactate-producing strain constructed.    Conclusions  This study applied CRISPR–Cas12a in  Z. mobilis  and established a genome editing tool for efficient and convenient genome engineering in  Z. mobilis  including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR–Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in  Z. mobilis  for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

Establishment and application of a CRISPR–Cas12a assisted genome-editing system in Zymomonas mobilis