Mappingin vivogenetic interactomics through Cpf1 crRNA array screening

Abstract

Genetic interactions lay the foundation of biological networks in virtually all organisms. Due to the complexity of mammalian genomes and cellular architectures, unbiased mapping of genetic interactions in vivo is challenging.Cpf1 is a single effector RNA-guided nuclease that enables multiplexed genome editing using crRNA arrays. Here we designed a Cpf1 crRNA array library targeting all pairwise permutations of the most significantly mutated nononcogenes, and performed double knockout screens in mice using a model of malignant transformation as well as a model of metastasis. CrRNA array sequencing revealed a quantitative landscape of all single and double knockouts.Enrichment, synergy and clonal analyses identified many unpredicted drivers and co-drivers of transformation and metastasis, with epigenetic factors as hubs of these highly connected networks. Our study demonstrates a powerful yet simple approach for in vivo mapping of unbiased genetic interactomes in mammalian species at a phenotypic level.

Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing

“…Cas12a nucleases, including AsCas12a and Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a), recognize target sites with T-rich protospacer adjacent motifs (PAMs) 1,2 , require a only single short ~40 nt CRISPR RNA (crRNA) to program target specificity 10 , and possess ribonuclease activity that enables multiplex targeting through poly-crRNA transcript processing 11 . Although Cas12a enzymes have shown utility for multiplex gene editing 12 , gene activation 13,14 , and combinatorial library screens 15 , one constraint is their requirement for a longer PAM of the form 5’-TTTV (where V is A, C, or G), which restricts targeting approximately six-fold relative to SpCas9. Although Cas12a orthologs from Francisella novicida (FnCas12a) and Moraxella bovoculi 237 (MbCas12a) were previously reported to recognize an increased number of PAMs in vitro 1 , our own findings (Supplementary Figs.…”

Cancer CRISPR Screens In Vivo

“…Like Cas9, the Cpf1 RGN can be used for precisely targeted genome editing, yet it has unique multiplexing capabilities due to its independence from a tracrRNA [64,65]. Cpf1 has just begun to emerge for higher-dimensional genetic screening of tumor growth and metastasis in vivo [66], and yet to be more broadly applied for other aspects of cancer. The development of this technology would enable high-dimensional screening of different mutation combinations, potentially leading to the identification of novel synergistic or synthetically lethal genetic interactions in cancer (Figure 3B).…”

Combinatorial CRISPR-Cas9 Metabolic Screens Reveal Critical Redox Control Points Dependent on the KEAP1-NRF2 Regulatory Axis

“…However, in the context of mammalian metabolism the SKO CRISPR approach comes with limitations, as redundancies and plasticity of the metabolic network may allow the system to remodel around a SKO, thereby confounding analyses of impact on cellular fitness. To overcome this challenge, our group and others recently developed combinatorial gene knockout screening approaches which may provide a more suitable platform to study gene dispensability and also systematically map their interactions (Boettcher et al, 2017; Chow et al, 2017; Han et al, 2017; Shen et al, 2017; Wong et al, 2016).…”