RT Journal Article
SR Electronic
T1 Selection-free non-viral method revealed highly efficient CRISPR-Cas9 genome editing of human pluripotent stem cells guided by cellular autophagy
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.05.16.444342
DO 10.1101/2021.05.16.444342
A1 Michelle Surma
A1 Kavitha Anbarasu
A1 Arupratan Das
YR 2021
UL http://biorxiv.org/content/early/2021/05/17/2021.05.16.444342.abstract
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.