Transgenic expression of enhanced AsCas12a in mice enables In Vivo multiplex genetic editing and development of diverse novel models of myeloid neoplasms

Abstract A central challenge for the development of engineered models of myeloid malignancies is the genetic complexity of human disease, as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) have an average of 4-5 recurrent mutations per patient. CRISPR-Cas9 can efficiently edit multiple genetic loci in murine hematopoietic stem cells (HSCs), but variability in editing multiple loci simultaneously can lead to heterogeneity in the resulting neoplasms. Enhanced AsCas12a (enAsCas12a) RNA-guided endonuclease enables multiplex editing of 4-5 genetic loci by processing an array of guide RNA (gRNA) expressed from a single vector. We generated a transgenic enAsCas12a mouse, enabling the consistent generation of myeloid malignancies with complex genetics. Methods To identify genetic combinations that lead to myeloid malignancies in mice, we first performed a CRISPR-Cas9 screen with a library of gRNAs targeting genes recurrently mutated in myeloid malignancies. Cas9+HSCs transduced with the gRNA library were transplanted into lethally irradiated mice. Primary and serially transplanted recipient bone marrow was analyzed by single cell RNA sequencing with gRNA capture (scRNA-seq). To generate models of myeloid malignancies with complex genetics, we generated Cas12a-mice by targeting C57/BL6 embryonic stem cells with a vector encoding the enAsCas12a cDNA, conditionally expressed by Cre-recombinase. Hematopoietic stem and progenitor cells (HSPCs) from VavCre;Cas12a-mice were transduced with a single viral vector with an array of up to four gRNA in series and transplanted into irradiated recipients. Results We first used CRISPR-Cas9 to identify genetic characteristics of myeloid malignancies generated by CRISPR technology. Serial transplantation of gRNA modified Cas9-HSC selected for aggressive myeloid malignancies, resulting in peripheral blasts and increased spleen size,. We characterized the malignant clones using scRNA-seq and identified the gRNA expressed by each cell. At quaternary stages, myeloid neoplasms were enriched for increased multiplicity of gRNA per cell: average unique gRNA per cell in Primary: 1.5 vs Quaternary Stage: 3.2; (Poisson model: p<0.001). However, the composition of genetic edits was heterogeneous and the resultant phenotype distinct to each experiment. We hypothesized CRISPR-Cas12 would improve the reproducibility of the disease models generated by increasing the uniformity of genetic edits in each cell. We generated a conditional Cas12a-mouse and confirmed enAsCas12a expression in HSCs. Neither peripheral blood counts nor composition of stem-progenitor compartments were altered by Cas12 expression. Functionally, mean editing efficiency across 10 genes recurrently mutated in human MDS/AML was 74% (S.D. 27.3%). As proof of concept, we transduced HSPCs from Cas12a-mice with either vectors encoding control, Nf1, Trp53 or both (Nf1-Trp53) gRNA. NGS demonstrated editing at the Nf1 and the Trp53 locus only in the HSPCs receiving the designated gRNA. Recipient mice only demonstrated disease in those with both Nf1-Trp53 gRNA, which was serially transplantable. Circulating pro-erythroblasts were present, pathognomonic of human acute erythroid leukemia, known to be enriched for TP53 mutations. To demonstrate the rapid applicability of this model, we contrasted Nf1-Trp53 to Nf1-Runx1 editedCas12a-HSPCs. The modification of Runx1 allele resulted in a myeloid differentiation bias, in comparison, replicated in analysis of human NF1 mutated MDS. We next designed gRNA arrays targeting four genes, focusing on the mutations found in MDS due to the paucity of murine models of this disease. As co-mutations in TET2-ZRSR2-EZH2-RUNX1 result in a poor prognosis MDS phenotype we generated a model of this disease by transducing Cas12a-murine HSPCs with a single vector encoding gRNA, targeting all four genes simultaneously. Analysis of gRNA targeted amplicon sequence from bone marrow confirmed editing at 4 genes. Tet2-Zrsr2-Ezh2-Runx1-mutant Cas12a-HSPCs resulted in block of megakaryocytic differentiation, with thrombocytopenia alongside anemia and evidence of erythroid dysplasia. Thus, we can generate multiple models of myeloid neoplasms from multiplexed genetic editing resulting in diverse and distinct disease phenotypes.Conclusion: We validate a novel Cas12a-mice which enables the efficient editing of multiple genes in HSPCs, advancing patient personalized models of myeloid neoplasms.