Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters.
The cloning of long DNA segments, especially those containing large gene clusters, is of particular importance to synthetic and chemical biology efforts for engineering organisms. While cloning has been a defining tool in molecular biology, the cloning of long genome segments has been challenging. Here we describe a technique that allows the targeted cloning of near-arbitrary, long bacterial genomic sequences of up to 100 kb to be accomplished in a single step. The target genome segment is excised from bacterial chromosomes in vitro by the RNA-guided Cas9 nuclease at two designated loci, and ligated to the cloning vector by Gibson assembly. This technique can be an effective molecular tool for the targeted cloning of large gene clusters that are often expensive to synthesize by gene synthesis or difficult to obtain directly by traditional PCR and restriction-enzyme-based methods.
School of Life Sciences, Center for Synthetic and Systems Biology, MOE Key Laboratory of Bioinformatics, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University, Beijing 100084, China.
Wenjun
W
Jiang
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Xuejin
X
Zhao
Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University, Tel Aviv 6997801, Israel.
Tslil
T
Gabrieli
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Chunbo
C
Lou
Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University, Tel Aviv 6997801, Israel.
Yuval
Y
Ebenstein
School of Life Sciences, Center for Synthetic and Systems Biology, MOE Key Laboratory of Bioinformatics, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University, Beijing 100084, China.
Ting F
TF
Zhu
D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins / Q000378:metabolism*; D000076987:CRISPR-Associated Protein 9; D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D016680:Genome, Bacterial / Q000235:genetics; D005810:Multigene Family / Q000235:genetics*; D013302:Streptomyces / Q000235:genetics; D013304:Streptomyces aureofaciens / Q000235:genetics; D014764:Viral Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases