Efficient and multiplexed somatic genome editing with Cas12a mice.

Somatic genome editing in mouse models has increased our understanding of the in vivo effects of genetic alterations in areas ranging from neuroscience to cancer biology and beyond. However, existing models are limited in their ability to create multiple targeted edits. Thus, our understanding of the complex genetic interactions that underlie development, homeostasis, and disease remains incomplete. Cas12a is an RNA-guided endonuclease with unique attributes that enable simple targeting of multiple genes with crRNA arrays containing tandem guides. To accelerate and expand the generation of complex genotypes in somatic cells, we generated transgenic mice with Cre-regulated and constitutive expression of enhanced Acidaminococcus sp. Cas12a (enAsCas12a). In these mice, enAsCas12a-mediated somatic genome editing robustly generated compound genotypes, as exemplified by the initiation of diverse cancer types driven by homozygous inactivation of trios of tumor suppressor genes. We further integrated these modular crRNA arrays with clonal barcoding to quantify the size and number of tumors with each array, as well as the efficiency of each crRNA. These Cas12a alleles will enable the rapid generation of disease models and broadly facilitate the high-throughput investigation of coincident genomic alterations in somatic cells in vivo .

The bromo-adjacent homology domains of PBRM1 associate with histone tails and contribute to PBAF-mediated gene regulation.

A critical component of gene regulation is recognition of histones and their post-translational modifications by transcription-associated proteins or complexes. Although many histone-binding reader modules have been characterized, the bromo-adjacent homology (BAH) domain family of readers is still poorly characterized. A pre-eminent member of this family is PBRM1 (BAF180), a component of the PBAF chromatin-remodeling complex. PBRM1 contains two adjacent BAH domains of unknown histone-binding potential. We evaluated the tandem BAH domains for their capacity to associate with histones and to contribute to PBAF-mediated gene regulation. The BAH1 and BAH2 domains of human PBRM1 broadly interacted with histone tails, but they showed a preference for unmodified N-termini of histones H3 and H4. Molecular modeling and comparison of the BAH1 and BAH2 domains with other BAH readers pointed to a conserved binding mode via an extended open pocket and, in general, an aromatic cage for histone lysine binding. Point mutants that were predicted to disrupt the interaction between the BAH domains and histones reduced histone binding in vitro and resulted in dysregulation of genes targeted by PBAF in cellulo. Although the BAH domains in PBRM1 were important for PBAF-mediated gene regulation, we found that overall chromatin targeting of PBRM1 was not dependent on BAH-histone interaction. Our findings identify a function of the PBRM1 BAH domains in PBAF activity that is likely mediated by histone tail interaction.