Advancing the genetic engineering toolbox by combiningAsCas12aknock-in mice with ultra-compact screening

Abstract

AbstractCas12a is a gene-editing tool that simplifies multiplexed gene targeting through its RNase activity, enabling maturation of individual crRNAs from a pre-crRNA-encoding RNA. Here, we present a mouse model that constitutively expresses enhancedAcidaminococcus sp. Cas12a(enAsCas12a) linked to an mCherry fluorescent reporter. We demonstrate efficient single and multiplexed gene-editing in cells fromenAsCas12aKImice. To testin vivoactivity, we transduced haematopoietic stem cells fromEμ-MycT/+;enAsCas12aKI/+animals withTrp53-targeting pre-crRNAs followed by transplantation into irradiated recipient animals. Tumour development was accelerated and TRP53 protein lost. We generated compact, genome-wide Cas12a knockout libraries targeting each gene with four guide RNAs encoded on two (Menuetto) or one (Scherzo) vector. Introducing these libraries intoEμ-MycT/+;enAsCas12aKI/+lymphoma cells followed by treatment with an MCL-1 inhibitor (S63845) or TRP53-inducer (nutlin-3a) identified known and novel drug resistance genes. Finally, we demonstrate simultaneous gene knockouts (Trp53 or combined Bax/Bak) and activation (Cd19) in primary T cells and mouse dermal fibroblasts from crosses of ourenAsCas12aand CRISPR activation models (dCas9a-SAM). OurenAsCas12amouse model and accompanying libraries enhance genome engineering capabilities and complements current CRISPR technologies.