초록
<P><B>Abstract</B></P> <P>Although CRISPR-Cas9/Cpf1 have been employed as powerful genome engineering tools, heterologous CRISPR-Cas9/Cpf1 are often difficult to introduce into bacteria and archaea due to their severe toxicity. Since most prokaryotes harbor native CRISPR-Cas systems, genome engineering can be achieved by harnessing these endogenous immune systems. Here, we report the exploitation of Type I-B CRISPR-Cas of <I>Clostridium tyrobutyricum</I> for genome engineering. <I>In silico</I> CRISPR array analysis and plasmid interference assay revealed that TCA or TCG at the 5′-end of the protospacer was the functional protospacer adjacent motif (PAM) for CRISPR targeting. With a lactose inducible promoter for CRISPR array expression, we significantly decreased the toxicity of CRISPR-Cas and enhanced the transformation efficiency, and successfully deleted <I>spo0A</I> with an editing efficiency of 100%. We further evaluated effects of the spacer length on genome editing efficiency. Interestingly, spacers ≤ 20 nt led to unsuccessful transformation consistently, likely due to severe off-target effects; while a spacer of 30–38 nt is most appropriate to ensure successful transformation and high genome editing efficiency. Moreover, multiplex genome editing for the deletion of <I>spo0A</I> and <I>pyrF</I> was achieved in a single transformation, with an editing efficiency of up to 100%. Finally, with the integration of the alcohol dehydrogenase gene (<I>adhE1</I> or <I>adhE2</I>) to replace <I>cat1</I> (the key gene responsible for butyrate production and previously could not be deleted), two mutants were created for n-butanol production, with the butanol titer reached historically record high of 26.2 g/L in a batch fermentation. Altogether, our results demonstrated the easy programmability and high efficiency of endogenous CRISPR-Cas. The developed protocol herein has a broader applicability to other prokaryotes containing endogenous CRISPR-Cas systems. <I>C. tyrobutyricum</I> could be employed as an excellent platform to be engineered for biofuel and biochemical production using the CRISPR-Cas based genome engineering toolkit.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The native Type I-B CRISPR-Cas of <I>C. tyrobutyricum</I> was harnessed for genome editing. </LI> <LI> Off-target effects were revealed, and appropriate spacer length was suggested. </LI> <LI> Single and multiplex genome editing were achieved with efficiencies of up to 100%. </LI> <LI> Carbon flow was redirected to butanol by replacement of <I>cat1</I> gene with <I>adhE1</I>/<I>adhE2</I>. </LI> <LI> High level butanol (26.2 g/L) was obtained in batch fermentation at low temperature. </LI> </UL> </P>