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

By Fillip Port, Simon L Bullock

Posted 31 Mar 2016
bioRxiv DOI: 10.1101/046417 (published DOI: 10.1038/nmeth.3972)

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 tRNAs, permits highly efficient multiplexing of Cas9-based mutagenesis. We also demonstrate that the tRNA-gRNA system markedly increases the efficacy of conditional gene disruption by Cas9 and can promote editing by the recently discovered RNA-guided endonuclease Cpf1.

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