Details

Autor(en) / Beteiligte

• Howden, Sara E.
• McColl, Bradley
• Glaser, Astrid
• Vadolas, Jim
• Petrou, Steven
• Little, Melissa H.
• Elefanty, Andrew G.
• Stanley, Edouard G.

Titel

A Cas9 Variant for Efficient Generation of Indel-Free Knockin or Gene-Corrected Human Pluripotent Stem Cells

Ist Teil von

• Stem cell reports, 2016-09, Vol.7 (3), p.508-517

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2016

Quelle

MEDLINE

Beschreibungen/Notizen

• While Cas9 nucleases permit rapid and efficient generation of gene-edited cell lines, the CRISPR-Cas9 system can introduce undesirable “on-target” mutations within the second allele of successfully modified cells via non-homologous end joining (NHEJ). To address this, we fused the Streptococcus pyogenes Cas9 (SpCas9) nuclease to a peptide derived from the human Geminin protein (SpCas9-Gem) to facilitate its degradation during the G1 phase of the cell cycle, when DNA repair by NHEJ predominates. We also use mRNA transfection to facilitate low and transient expression of modified and unmodified versions of Cas9. Although the frequency of homologous recombination was similar for SpCas9-Gem and SpCas9, we observed a marked reduction in the capacity for SpCas9-Gem to induce NHEJ-mediated indels at the target locus. Moreover, in contrast to native SpCas9, we demonstrate that transient SpCas9-Gem expression enables reliable generation of both knockin reporter cell lines and genetically repaired patient-specific induced pluripotent stem cell lines free of unwanted mutations at the targeted locus. •“On-target” deleterious mutations are often encountered with CRISPR-Cas9 gene editing•SpCas9-Gem retains gene-targeting efficiency with reduced NHEJ activity•SpCas9-Gem facilitates derivation of “indel-free” gene-edited iPSCs The use of the Cas9/CRISPR system for efficient generation of precisely modified human pluripotent stem cells without secondary deleterious mutations in the untargeted allele can be challenging. In this article, Howden and colleagues describe a variant of Cas9 that has been fused to a peptide derived from human Geminin to facilitate its degradation during G1 phase of the cell cycle when DNA repair by NHEJ predominates. Using this variant (SpCas9-Gem) they demonstrate reliable and efficient derivation of both knockin reporter iPSCs and genetically repaired patient-specific iPSC lines free of NHEJ-mediated indels at the target locus.