Expansion of the CRISPR toolbox in an animal with tRNA-flanked Cas9 and Cpf1 gRNAs

Abstract

We present vectors for producing multiple CRISPR gRNAs from a single RNA polymerase II or III transcript in Drosophila. The system, which is based on liberation of gRNAs by processing of flanking t {dagger}RNAs, permits highly efficient multiplexing of Cas9-based mutagenesis. We also demonstrate that the {dagger}RNA-gRNA system markedly increases the efficacy of conditional gene disruption by Cas9 and can promote editing by the recently discovered RNA-guided endonuclease Cpf1.