RESUMO
Systematic investigation of the genetic interactions that influence metastatic potential has been challenging. Here we developed massively parallel CRISPR-Cpf1/Cas12a crRNA array profiling (MCAP), an approach for combinatorial interrogation of double knockouts in vivo. We designed an MCAP library of 11,934 arrays targeting 325 pairwise combinations of genes implicated in metastasis. By assessing the metastatic potential of the double knockouts in mice, we unveiled a quantitative landscape of genetic interactions that drive metastasis.
Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Endonucleases/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Metástase Neoplásica/genética , Animais , Proteína 9 Associada à CRISPR/genética , Linhagem Celular Tumoral , Camundongos , Análise de Sequência de RNARESUMO
Mutations and genetic alterations are often sequentially acquired in various biological and pathological processes, such as development, evolution, and cancer. Certain phenotypes only manifest with precise temporal sequences of genetic events. While multiple approaches have been developed to model the effects of mutations in tumorigenesis, few recapitulate the stepwise nature of cancer evolution. Here we describe a flexible sequential mutagenesis system, Cpf1-Flip, with inducible inversion of a single crRNA array (FlipArray), and demonstrate its application in stepwise mutagenesis in murine and human cells. As a proof-of-concept, we further utilize Cpf1-Flip in a pooled-library approach to model the acquisition of diverse resistance mutations to cancer immunotherapy. Cpf1-Flip offers a simple, versatile, and controlled approach for precise mutagenesis of multiple loci in a sequential manner.