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

Wei Jin, Yexuan Deng, John E. La Marca, Emily J. Lelliott, Sarah T. Diepstraten, Valentina Snetkova, Kristel M. Dorighi, Luke Hoberecht, Lauren Whelan, Yang Liao, Lin Tai, Geraldine Healey, Wei Shi, Andrew J. Kueh, Benjamin Haley, Jean-Philippe Fortin, Marco J. Herold

crossref(2024)

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摘要
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 enAsCas12aKI mice. To test in vivo activity, we transduced haematopoietic stem cells from E μ -MycT/+;enAsCas12aKI/+ 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 -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 our enAsCas12a and CRISPR activation models ( dCas9a-SAM ). Our enAsCas12a mouse model and accompanying libraries enhance genome engineering capabilities and complements current CRISPR technologies. ### Competing Interest Statement KMD, LH, and JPF are current employees of Genentech. BH and VS have previously been employees of Genentech.
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