%0 Journal Article %A Darin Bloemberg %A Daniela Sosa-Miranda %A Tina Nguyen %A Risini D. Weeratna %A Scott McComb %T Self-cutting and integrating CRISPR plasmids (SCIPs) enable targeted genomic integration of large genetic payloads for rapid cell engineering %D 2020 %R 10.1101/2020.03.25.008276 %J bioRxiv %P 2020.03.25.008276 %X 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 efficiency and precision of the payload but did not affect integration of the remaining plasmid sequence. As proofs-of-concept, we used SCIPay to 1) insert a gene fragment encoding tdTomato into the CD69 locus of Jurkat cells, thereby creating a NOVEL cell line that reports T cell activation, and 2) insert a chimeric antigen receptor (CAR) gene into the T cell receptor alpha constant (TRAC) locus. Here, we demonstrate that SCIPs function as simple, efficient, and programmable tools useful for generating gene knockout/knock-in cell lines and suggest future utility in knock-in site screening/optimization, unbiased off-target site identification, and multiplexed, iterative, and/or library-scale automated genome engineering.Contribution to the Field We present here the first report of a novel, straightforward, and highly time- and cost-efficient system for targeted delivery of large synthetic DNA payloads into almost any desired site within a mammalian genome. By including a self-cleaving site within a well-known CRISPR plasmid, this single-component platform enables genomic integration by simple plasmid transfection and is easily re-programmable. Introducing self-cleaving plasmids to human and mouse cell lines produces similarly efficient gene knockout as non-cleaving plasmids, but results in stable integration of whole-plasmid DNA sequences into targeted genomic sites within a practical proportion of cells (up to 6%) which can then be selected and expanded. We demonstrate how self-cleaving and integrating plasmids (SCIP) can be modified to deliver specific transgenes to targeted locations by recombining sgRNA sequences with more than one self-cleavage site. We believe that pSCIP represents a valuable cell engineering tool that is much simpler to use and modify than existing genome engineering techniques involving in vitro DNA synthesis, virus handling and production, or expensive synthesis of long single-stranded DNA repair templates. %U https://www.biorxiv.org/content/biorxiv/early/2020/03/25/2020.03.25.008276.full.pdf