Abstract
CRISPR/Cas9 technology enables efficient, rapid and cost-effective targeted genomic modification in a wide variety of cellular contexts including cultured cells. Some applications such as generation of double knock-outs, large deletions and paired-nickase cleavage require simultaneous expression of two gRNAs. Although single plasmids that enable multiplex expression of gRNAs have been developed, these require multiple rounds of cloning and/or PCR for generation of the desired construct. Here, we describe a series of vectors that enable generation of customized dual-gRNA expression constructs via an easy one-step golden gate cloning reaction using two annealed oligonucleotide inserts with different overhangs. Through nucleofection of mouse embryonic stem cells, we demonstrate highly efficient cleavage of the target loci using the dual-guide plasmids, which are available as Cas9-nuclease or Cas9-nickase expression constructs, with or without selection markers. These vectors are a valuable addition to the CRISPR/Cas9 toolbox and will be made available to all interested researchers via the Addgene plasmid repository.
Highlights
CRISPR/Cas9 technology is a powerful genome editing tool that has become widely used by researchers to generate targeted genetic modifications in many contexts including cultured cell lines and zygotes
The second cassette was positioned in the opposite orientation to the original hU6-guide RNA (gRNA) expression cassette to reduce the possibility of recombination (Fig 1A)
Plasmids from the Zhang laboratory have greatly simplified generation of customized gRNACas9/Cas9-nickase expression constructs through utilization of the golden gate cloning strategy
Summary
CRISPR/Cas technology is a powerful genome editing tool that has become widely used by researchers to generate targeted genetic modifications in many contexts including cultured cell lines and zygotes. CRISPR/Cas offers several advantages over preexisting genome editing technologies including ease of use, relatively low cost and high activity [1,2,3,4,5]. Generation of a targeted DSB can be achieved by delivery of Cas and gRNA components in plasmid, RNA or ribonucleoprotein (RNP) forms. For some applications, such as cultured cells, plasmids are generally preferred due to their ease of generation and stability.
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