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

Clicks: 308
ID: 104970
2020
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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.
Reference Key
leung2020rnabasedjournal Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors Leung, Alan W;Broton, Cayla;Bogacheva, Mariia S;Xiao, Andrew Z;Garcia-Castro, Martin I;Lou, Yan-Ru;
Journal Journal of molecular biology
Year 2020
DOI
S0022-2836(20)30305-3
URL
Keywords
transfection cas9 messenger rna clustered regularly interspaced short palindromic repeats lipofection crrna:tracrrna duplex

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