Programmable sequential mutagenesis by inducible Cpf1 crRNA array inversion

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-ofconcept, 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.