Efficient Genome Editing in Clostridium cellulolyticum via CRISPR-Cas9 Nickase.
The CRISPR-Cas9 system is a powerful and revolutionary genome-editing tool for eukaryotic genomes, but its use in bacterial genomes is very limited. Here, we investigated the use of the Streptococcus pyogenes CRISPR-Cas9 system in editing the genome of Clostridium cellulolyticum, a model microorganism for bioenergy research. Wild-type Cas9-induced double-strand breaks were lethal to C. cellulolyticum due to the minimal expression of nonhomologous end joining (NHEJ) components in this strain. To circumvent this lethality, Cas9 nickase was applied to develop a single-nick-triggered homologous recombination strategy, which allows precise one-step editing at intended genomic loci by transforming a single vector. This strategy has a high editing efficiency (>95%) even using short homologous arms (0.2 kb), is able to deliver foreign genes into the genome in a single step without a marker, enables precise editing even at two very similar target sites differing by two bases preceding the seed region, and has a very high target site density (median interval distance of 9 bp and 95.7% gene coverage in C. cellulolyticum). Together, these results establish a simple and robust methodology for genome editing in NHEJ-ineffective prokaryotes.
Institute for Environmental Genomics and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma, USA.
Tao
T
Xu
Institute for Environmental Genomics and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma, USA.
Yongchao
Y
Li
Institute for Environmental Genomics and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma, USA.
Zhou
Z
Shi
Institute for Environmental Genomics and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma, USA.
Christopher L
CL
Hemme
Institute for Environmental Genomics and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma, USA.
Yuan
Y
Li
College of Biology, Hunan University, Changsha, China.
Yonghua
Y
Zhu
Institute for Environmental Genomics and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma, USA.
Joy D
JD
Van Nostrand
Institute for Environmental Genomics and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma, USA.
Zhili
Z
He
Institute for Environmental Genomics and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma, USA Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA State Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, Tsinghua University, Beijing, China jzhou@ou.edu.
Jizhong
J
Zhou
D064113:CRISPR-Cas Systems; D046973:Ruminiclostridium cellulolyticum / Q000201:enzymology* / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D005827:Genetics, Microbial / Q000379:methods*; D059765:Homologous Recombination; D008967:Molecular Biology / Q000379:methods*; D013297:Streptococcus pyogenes / Q000201:enzymology; D014169:Transformation, Bacterial
D003850:Deoxyribonuclease I