Cloning-free CRISPR
2015
We present self-cloning
CRISPR/
Cas9(scCRISPR), a technology that allows for
CRISPR/
Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-
guide RNA(sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving
palindromicsgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through
homologous recombination. scCRISPR enables efficient generation of
gene knockouts(∼88%
mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%–4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human
transgenesis.
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