An episomal vector-based CRISPR/Cas9 system for highly efficient gene knockout in human pluripotent stem cells

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.

A compact Cas9 ortholog from Staphylococcus Auricularis (SauriCas9) expands the DNA targeting scope

“…The RNA-guided CRISPR/Cas9 system is a powerful tool for genome editing in diverse organisms and cell types [1][2][3][4][5]. In this system, a Cas9 nuclease and a guide RNA (gRNA) form a Cas9-gRNA complex, which recognizes a gRNA complementary DNA sequence and generates a site-specific double-strand break (DSB) [1,2,6].…”

Scarless Genome Editing of Human Pluripotent Stem Cells via Transient Puromycin Selection

“…Generating a parental cell line with inducible or constitutive Cas9 expression has been shown to yield NHEJ efficiencies of up to 60% and precise HDR efficiencies up to 40% ( González et al., 2014 , Liang et al., 2017 ). A limitation of these methods is that the Cas9 construct is either permanently integrated ( González et al., 2014 , Cao et al., 2016 ) or must be later removed with a subsequent reagent delivery and/or clonal selection step ( Wang et al., 2017 , Xie et al., 2017 ) to achieve scarless editing. Alternatively, non-integrating methods rely on enrichment of cells transiently expressing Cas9 through use of a selectable marker ( Ding et al., 2013 , Ran et al., 2013 ).…”

Discovery and engineering of small SlugCas9 with broad targeting range and high specificity and activity

“…The active complex is composed of a Cas9 nuclease and a guide RNA (gRNA), which together recognize a target DNA that is complementary to the 20 bp-protospacer sequence in the gRNA. Upon recognition, this complex generates a site-specific doublestrand break (DSB) [5][6][7][8][9][10] . Target site recognition also requires a specific protospacer adjacent motif (PAM) 5,[11][12][13] unique to the Cas9 protein, which limits the targeting scope of Cas9.…”