Mar 26, 2020

Self-cutting and integrating CRISPR plasmids (SCIPs) enable targeted genomic integration of large genetic payloads for rapid cell engineering

BioRxiv : the Preprint Server for Biology
D. Bloemberg, D. Sosa-Miranda

Abstract

Since observations that CRISPR nucleases function in mammalian cells, many strategies have been devised to adapt them for genetic engineering. Here, we investigated self-cutting and integrating Cas9/CRISPR plasmids (SCIPs) as easy-to-use gene editing tools that insert themselves at CRISPR-guided locations. Leaky sgRNA/Cas9 expression in bacteria initially prevented SCIP assembly and production; this was ameliorated by inserting a mammalian intron into the Cas9 gene. SCIPs demonstrated similar expression kinetics and gene disruption efficiency in mouse (EL4) and human (Jurkat) cells, with stable integration in 3-6% of transfected cells. Clonal sequencing analysis indicated that integrants showed bi- or mono-allelic integration of entire CRISPR plasmids in predictable orientations and with limited indel formation. Interestingly, including longer homology arms (HAs) (500 bp) in varying orientations only modestly increased knock-in efficiency (~2-fold), indicating that SCIP integration favours homology-independent mechanisms. Using a SCIP-payload design (SCIPay) which liberates a promoter-less sequence flanked by HAs thereby requiring perfect homology-directed repair (HDR) for expression, longer HAs resulted in higher integration e...Continue Reading

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