title: CRISPR/Cas9 nuclease cleavage combined with Gibson assembly for seamless cloning.
abstract: Restriction enzymes have two major limitations for cloning: they cannot cleave at any desired location in a DNA sequence and may not cleave uniquely within a DNA sequence. In contrast, the clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme 9 (Cas9), when coupled with single guide RNAs (sgRNA), has been used in vivo to cleave the genomes of many species at a single site, enabling generation of mutated cell lines and animals. The Cas9/sgRNA complex recognizes a 17-20 base target site, which can be of any sequence as long as it is located 5' of the protospacer adjacent motif (PAM; sequence 5'-NRG, where R = G or A). Thus, it can be programmed to cleave almost anywhere with a stringency higher than that of one cleavage in a sequence of human genome size. Here, the Cas9 enzyme and a specific sgRNA were used to linearize a 22 kb plasmid in vitro. A DNA fragment was then inserted into the linearized vector seamlessly through Gibson assembly. Our technique can be used to directly, and seamlessly, clone fragments into vectors of any size as well as to modify existing constructs where no other methods are available.
authors: Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FL.
authors: Jia-Wang
authors: JW
authors: Wang
authors: Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FL.
authors: Amy
authors: A
authors: Wang
authors: Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FL.
authors: Kunyu
authors: K
authors: Li
authors: Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FL.
authors: Bangmei
authors: B
authors: Wang
authors: Department of Pediatrics, Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center and the University of Pittsburgh School of Medicine, Pittsburgh, PA.
authors: Shunqian
authors: S
authors: Jin
authors: Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FL.
authors: Michelle
authors: M
authors: Reiser
authors: Division of Allergy and Immunology, Department of Internal Medicine, University of South Florida Morsani College of Medicine, Tampa, FL.|James A. Haley Veterans' Hospital, Tampa, FL.
authors: Richard F
authors: RF
authors: Lockey
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D000076987:CRISPR-Associated Protein 9; D064130:CRISPR-Associated Proteins / Q000737:chemistry*; D064113:CRISPR-Cas Systems; D003001:Cloning, Molecular / Q000379:methods*; D053837:DNA Cleavage; D004720:Endonucleases / Q000737:chemistry*; D016678:Genome; D051379:Mice; D010957:Plasmids / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; C498954:Lrba protein, mouse; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: CRISPR cleavage; Cas9; Gibson cloning; large vector; single guide RNA