Abstract Cas12a 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 enhanced Acidaminococcus sp. Cas12a ( enAsCas12a ) linked to an mCherry fluorescent reporter. We demonstrate efficient single and multiplexed gene-editing in cells from enAsCas12a KI mice. To test in vivo activity, we transduced haematopoietic stem cells from E μ -Myc T/+ ;enAsCas12a KI/+ animals with Trp53 -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 into E μ -Myc T/+ ;enAsCas12a KI/+ 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 our enAsCas12a and CRISPR activation models ( dCas9a-SAM ). Our enAsCas12a mouse model and accompanying libraries enhance genome engineering capabilities and complements current CRISPR technologies.

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