Abstract
Human pluripotent stem cells (hPSCs) represent a unique opportunity for understanding the molecular mechanisms underlying complex traits and diseases. CRISPR/Cas9 is a powerful tool to introduce genetic mutations into the hPSCs for loss-of-function studies. Here, we developed an episomal vector-based CRISPR/Cas9 system, which we called epiCRISPR, for highly efficient gene knockout in hPSCs. The epiCRISPR system enables generation of up to 100% Insertion/Deletion (indel) rates. In addition, the epiCRISPR system enables efficient double-gene knockout and genomic deletion. To minimize off-target cleavage, we combined the episomal vector technology with double-nicking strategy and recent developed high fidelity Cas9. Thus the epiCRISPR system offers a highly efficient platform for genetic analysis in hPSCs.
Highlights
Human pluripotent stem cells represent a unique opportunity for understanding the molecular mechanisms underlying complex traits and diseases
We analyzed the capacity of the epiCRISPR system for supporting gene expression in Human pluripotent stem cells (hPSCs) (all the experiments in this study were performed in one human embryonic stem cells (hESCs) line (H9) and two induced pluripotent stem cells (iPSCs) lines)
We have developed an epiCRISPR system for highly efficient genome editing in hPSCs
Summary
Human pluripotent stem cells (hPSCs) represent a unique opportunity for understanding the molecular mechanisms underlying complex traits and diseases. Clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/ Cas9), adapted from microbial adaptive immune defense system[5, 6], is a recently reported genomic editing tool that is rapidly gaining popularity due to the ease of assembly and high efficiency[3, 4] In this system, a 20 nt guide RNA (gRNA) directs the Cas[9] nuclease to generate site-specific double-stranded breaks (DSBs). We used episomal CRISPR/Cas[9] system for efficient genome editing in hPSCs. The vector contains OriP/EBNA1 components originated from Epstein-Bar virus, which can drive plasmid duplication once per cell division in eukaryotes[24], allowing Cas[9] and gRNAs persistently expressed in cells; the vector contains a puromycin resistance gene, allowing enrichment of transfected cells by drug selection. The epiCRISPR system enables up to 100% gene knockout, which could facilitate efficient genetic analysis in hPSCs
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