Open AccessCRISPR/Cas9 nuclease cleavage combined with Gibson assembly for seamless cloning
Author(s) -
Jiawang Wang,
Amy Wang,
Kunyu Li,
Bangmei Wang,
Shunqian Jin,
Michelle Reiser,
Richard F. Lockey
Publication year - 2015
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/000114261
Subject(s) - crispr , cas9 , nuclease , cleave , genome editing , biology , plasmid , multiple cloning site , dna , restriction enzyme , computational biology , genetics , microbiology and biotechnology , gene , recombinant dna , expression vector
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.