Cloning-free CRISPR
Name
Arbab-2015-Cloning-free CRISPR.pdf
Size
1.44 MB
Format
Adobe PDF
Checksum (MD5)
77ba8b10f84cf771b5cf03956509d276
Author(s)
Arbab, Mandana
Srinivasan, Sharanya
Geijsen, Niels
Sherwood, Richard I.
Hashimoto, Tatsunori Benjamin
Date Issued
October 2015
Journal
Stem Cell Reports
Publisher
Elsevier
Citation
Arbab, Mandana, Sharanya Srinivasan, Tatsunori Hashimoto, Niels Geijsen, and Richard I. Sherwood. “Cloning-Free CRISPR.” Stem Cell Reports 5, no. 5 (November 2015): 908–917.
Version
Final published version
Abstract
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 palindromic sgRNA 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.
MIT Department
Massachusetts Institute of Technology. Computer Science and Artificial Intelligence Laboratory
Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science
Terms of Use
Creative Commons Attribution
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1016/j.stemcr.2015.09.022
