Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening

Cas12a is a next-generation gene-editing tool that enables multiplexed gene targeting. Here, we present a mouse model that constitutively expresses enhanced Acidaminococcus sp. Cas12a (enAsCas12a) linked to an mCherry fluorescent reporter. We demonstrate efficient single and multiplexed gene-editing in vitro, using primary and transformed cells from enAsCas12a mice. We further demonstrate successful in vivo gene-editing, using normal and cancer-prone enAsCas12a stem cells to reconstitute the haematopoietic system of wild-type mice. We also present compact, genome-wide Cas12a knockout libraries, with four crRNAs per gene encoded across one (Scherzo) or two (Menuetto) vectors, and demonstrate the utility of these libraries across methodologies: in vitro enrichment and drop-out screening in lymphoma cells and immortalised fibroblasts, respectively, and in vivo screens to identify lymphoma-driving events. Finally, we demonstrate CRISPR multiplexing via simultaneous gene knockout (via Cas12a) and activation (via dCas9-SAM) using primary T cells and fibroblasts. Our enAsCas12a mouse and accompanying crRNA libraries enhance genome engineering capabilities and complement current CRISPR technologies. Overall design: NGS of mouse cells containing CRISPR/Cas12 libraries (Menuetto/dual and Scherzo/quad). Sequences are of the sgRNA plasmids. Three screens were performed: 1) enrichment screen in mouse EuMyc lymphoma cells using S63845 and Nutlin-3a as selection pressures, 2) drop out screen in immortalised mouse dermal fibroblasts (MDFs), and 3) in vivo enrichment screen for drivers of lymphomagenesis in mice.