CRISPR-Cas12a-mediated Gene Deletion And Regulation In Clostridium Ljungdahlii And Its Application In Carbon Flux Redirection By Synthesis Gas Fermentation

Clustered regular interspaced short palindromic repeats(CRISPR)exist in the genomes of most bacteria and almost all archaea.In the vicinity of the CRISPR sequences,there are some genes coding CRISPR-associated proteins(called Cas proteins)that possess the function of endonuclease.Cas proteins,combined with the transcribed RNA of CRISPR,from CRISPR-Cas systems,which can be used by the hosts to defend against foreign viruses and phage invasion.Due to the activity of specific site cleavage of nucleic acids,CRISPR-Cas systems have been developed into genome editing tools in plants,animals,and microorganisms.Cas12a(also known as Cpfl),belonging to the Class ? type ? protein of the Cas protein family,contains a Ruvc domain and a Nuc domain.Cas12a has both DNA nuclease and RNA nuclease activity.Compared with Cas9 proteins,Cas 12a proteins cleave not only the target DNA,but also their own CRISPR transcriptional RNA precursor,thereby forming a mature crRNA(CRISPR RNA).Moreover,Cas 12a proteins normally contain approximate 100 amino acids,and thus are much smaller than Cas9 proteins.Moreover,the sequence of PAM(protospacer adjacent motif)recognized by Cas12a proteins is normally 5'-NGG-3' that recognized by Cas9 proteins.Thus,Casl2a proteins are more suitable than Cas9 proteins to be used in the CRISPR-Cas-based genome editing of AT-rich bacteriaClostridium ljungdahlii is one of the representative strains of acetogenic species.This strain has the Wood-Ljungdahl carbon sequestration pathway,which can use CO/C02 gas as the sole carbon source to produce organic acids and alcohols,exhibiting promising application values.However,as a low-GC content Gram-positive bacterium,the GC content in the whole genome of C.ljungdahlii is only 31%Therefore,PAM sequences available for the CRISPR-Cas9 system are very limited For some sequences,especially the short ones which lacking the 5'-NGG-3' PAM site could not be edited by the CRISPR-Cas9 system.In the contrary,Cas 12a proteins recognize T-rich PAM sequences,and therefore,developing the CRISPR-Cas 12a-based genome editing tools are of great significance for Clostridium ljungdahlii,which can effectively improve editing efficiency and flexibility,and make up for the defects and deficiencies of the original CRISPR-Cas9 systemIn this study,we established the CRISPR-Cas12a-based gene editing tool in C.ljungdahlii,achieving both gene knockout and gene expression interference.We first tested Cas12a proteins from four different sources,and fortunately,found that the Cas12a protein from Francisella tularensis subsp.novicida U112 showed no obvious toxicity to Clostridium ljungdahlii.Then,FnCas12a was proven to has the activity of cutting double-strand DNA in vivo.Based on this,we established the CRISPR-Cas12a-based gene knockout method in C.ljungdahlii,assisted by an improved electroporation method.Four genes were successfully knocked out,namely pta(CLJU_c12770,encoding phosphoacetyltransferase),adhEl(CLJU_c16510,encoding bifunctional aldehyde/alcohol dehydrogenase),ctf(CLJU c39430,encoding acyl-CoA transferase)and pyre(CLJU_c35680,encoding orotate phosphoribosyltransferase).In addition,a DNase-deactivated FnCasl2a(ddFnCasl2a)that had an E1006A mutation in the RuvC domain was adopted.The CRISPRi system based on ddFnCasl2a was built up,and we chose the four genes in the fructose culture condition for test,they are CLJU_c09110(the gene encoding the anaerobic carbon monoxide dehydrogenase catalytic subunit),CLJU_c04990(the gene encoding the surface protein),CLJU c37650(a gene encoding formic acid-tetrahydrofolate ligase)and CLJU cl6510(a gene encoding a bifunctional aldehyde/alcohol dehydrogenase).The highest repression degree for CLJU c09110,CLJU c04990,CLJU c37650 and CLJU_c16510 was 99%,96%,88%,97%,respectively.These data suggest that the crRNA-binding positions around the translation initiation site are suitable for CRISPRi manipulations in C.ljungdahlii.Based on the findings,we chose the pta gene in C.ljungdahlii for transcriptional repression to examine whether expected phenotypic changes occurred.That pat is essential and located in the acetic acid-forming pathway,which represents a major ATP donor during gas fermentation in C.ljungdahlii.Thus,the inactivation of pta impaird cell growth and acetate production.Having established Cas12a-mediated CRISPRi in C.ljungdahlii,we next sought to investigate the utility of this tool for regulating carbon flow in product synthesis in this anaerobe.An engineered C.ljungdahlii strain in which a BA-producing pathway derived from C.acetobutylicum has been integrated into chromosome was chosen for a test.Through CRISPR-ddFnCas system,the expression of the adhEl gene was inhibited in the engineered strain integrating the butyric acid pathway,and the redistribution of carbon flux was achieved.According to the total solvent ratio,the distribution of ethanol,acetic acid and butyric acid were all distributed.The ethanol ratio decreased from 4.6%(control)to 2.6%,the butyric acid ratio increased from 26.8%(control)to 32.1%,and the yield of butyric acid increased by 20%compared to the control group.This study further expands the molecular genetic manipulation toolbox of C.ljungdahlii,providing more powerful supports for basic and application researches of this important anaerobic bacterium.This new tool also fills a gap in the existing gene editing toolbox of C ljungdahlii and will greatly assist in the discovery of essential and functional genes associated with important physiological and metabolic processes at the genome level.