Bacterial Genome Editing with CRISPR-Cas9: Deletion, Integration, Single Nucleotide Modification, and Desirable "Clean" Mutant Selection in Clostridium beijerinckii as an Example.
CRISPR-Cas9 has been demonstrated as a transformative genome engineering tool for many eukaryotic organisms; however, its utilization in bacteria remains limited and ineffective. Here we explored Streptococcus pyogenes CRISPR-Cas9 for genome editing in Clostridium beijerinckii (industrially significant but notorious for being difficult to metabolically engineer) as a representative attempt to explore CRISPR-Cas9 for genome editing in microorganisms that previously lacked sufficient genetic tools. By combining inducible expression of Cas9 and plasmid-borne editing templates, we successfully achieved gene deletion and integration with high efficiency in single steps. We further achieved single nucleotide modification by applying innovative two-step approaches, which do not rely on availability of Protospacer Adjacent Motif sequences. Severe vector integration events were observed during the genome engineering process, which is likely difficult to avoid but has never been reported by other researchers for the bacterial genome engineering based on homologous recombination with plasmid-borne editing templates. We then further successfully employed CRISPR-Cas9 as an efficient tool for selecting desirable "clean" mutants in this study. The approaches we developed are broadly applicable and will open the way for precise genome editing in diverse microorganisms.
Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Yi
Y
Wang
Department of Chemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Zhong-Tian
ZT
Zhang
Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Seung-Oh
SO
Seo
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Patrick
P
Lynn
Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.|Department of Bioengineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Ting
T
Lu
Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Yong-Su
YS
Jin
Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.|The Integrated Bioprocessing Research Laboratory (IBRL), University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Hans P
HP
Blaschek
D064113:CRISPR-Cas Systems; D046970:Clostridium beijerinckii / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D017353:Gene Deletion; D000072669:Gene Editing; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D016680:Genome, Bacterial; D059765:Homologous Recombination; D009711:Nucleotides / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D000094704:RNA, Guide, CRISPR-Cas Systems
D009711:Nucleotides; D000094704:RNA, Guide, CRISPR-Cas Systems
CRISPR-Cas9; genome engineering; homologous recombination; single nucleotide modification (SNM); synthetic biology; vector integration event (VIE)