PT  - JOURNAL ARTICLE
AU  - Michelle Surma
AU  - Kavitha Anbarasu
AU  - Arupratan Das
TI  - Selection-free non-viral method revealed highly efficient CRISPR-Cas9 genome editing of human pluripotent stem cells guided by cellular autophagy
AID  - 10.1101/2021.05.16.444342
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.05.16.444342
4099  - http://biorxiv.org/content/early/2021/05/17/2021.05.16.444342.short
4100  - http://biorxiv.org/content/early/2021/05/17/2021.05.16.444342.full
AB  - CRISPR-Cas9 mediated genome editing of human pluripotent stem cells (hPSCs) provides strong avenues for human disease modeling, drug discovery and cell replacement therapy. Genome editing of hPSCs is an extremely inefficient process and requires complex gene delivery and selection methods to improve edit efficiency which are not ideal for clinical applications. Here, we have shown a selection free simple lipofectamine based transfection method where a single plasmid encoding guide RNA (gRNA) and Cas9 selectively transfected hPSCs at the colony edges. Upon dissection and sequencing, the edge cells showed more than 30% edit frequency compared to the reported 3% rate under no selections. Increased cellular health of the edge cells as revealed by reduced autophagy gene-expressions is critical for such transfection pattern. Edge specific transfection was inhibited by blocking lysosomal activity which is essential for autophagy. Hence, our method provides robust scarless genome-editing of hPSCs which is ideal for translational research.Competing Interest StatementThe authors have declared no competing interest.