Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs

Expressing guide RNAs from a tRNA scaffold enhances mutagenesis by both Cas9 and Cpf1 and allows conditional CRISPR applications in vivo. We present tRNA-based vectors for producing multiple clustered regularly interspaced short palindromic repeats (CRISPR) single guide RNAs (sgRNAs) from a single RNA polymerase II or III transcript in Drosophila. The system, which is based on liberation of sgRNAs by processing flanking tRNAs, permits highly efficient multiplexing of Cas9-based mutagenesis. We also demonstrate that the tRNA–sgRNA system markedly increases the efficacy of conditional gene disruption by Cas9 and can promote editing by the recently discovered RNA-guided endonuclease Cpf1.