RNA-based CRISPR-Mediated Loss-of-Function Mutagenesis in Human Pluripotent Stem Cells

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Leung , A W , Broton , C , Bogacheva , M S , Xiao , A Z , Garcia-Castro , M I & Lou , Y-R 2020 , ' RNA-based CRISPR-Mediated Loss-of-Function Mutagenesis in Human Pluripotent Stem Cells ' , Journal of Molecular Biology , vol. 432 , no. 13 , pp. 3956-3964 . https://doi.org/10.1016/j.jmb.2020.04.017

Title: RNA-based CRISPR-Mediated Loss-of-Function Mutagenesis in Human Pluripotent Stem Cells
Author: Leung, Alan W.; Broton, Cayla; Bogacheva, Mariia S.; Xiao, Andrew Z.; Garcia-Castro, Martin I.; Lou, Yan-Ru
Contributor organization: Division of Pharmaceutical Biosciences
Tissue engineering for drug research
Drug Research Program
Date: 2020-06-12
Language: eng
Number of pages: 9
Belongs to series: Journal of Molecular Biology
ISSN: 0022-2836
DOI: https://doi.org/10.1016/j.jmb.2020.04.017
URI: http://hdl.handle.net/10138/329373
Abstract: Current approaches for Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-Associated-9 (Cas9)-mediated genome editing in human pluripotent stem (PS) cells mainly employ plasmids or ribonucleoprotein complexes. Here, we devise an improved transfection protocol of in vitro transcribed Cas9 mRNA and crRNA:tracrRNA duplex that can effectively generate indels in four genetic loci (two active and two inactive) and demonstrate utility in four human PS cell lines (one embryonic and three induced PS cell lines). Our improved protocol incorporating a Cas9-linked selection marker and a staggered transfection strategy promotes targeting efficiency up to 85% and biallelic targeting efficiency up to 76.5% of total mutant clones. The superior targeting efficiency and the non-integrative nature of our approach underscores broader applications in high-throughput arrayed CRISPR screening and in generating custom-made or off-the-shelf cell products for human therapy.
Cas9 messenger RNA
clustered regularly interspaced short palindromic repeats
crRNA:tracrRNA duplex
1182 Biochemistry, cell and molecular biology
318 Medical biotechnology
Peer reviewed: Yes
Rights: cc_by_nc_nd
Usage restriction: openAccess
Self-archived version: acceptedVersion

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