Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Animals, Genetically Modified / genetics
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CRISPR-Associated Proteins / genetics*
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CRISPR-Cas Systems / genetics
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Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
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Drosophila / enzymology
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Drosophila / genetics*
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Mutagenesis, Site-Directed
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Plasmids
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RNA Editing / genetics
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RNA Polymerase II / genetics
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RNA Polymerase III / genetics
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RNA, Guide, CRISPR-Cas Systems / genetics*
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RNA, Transfer / genetics*
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Transcription, Genetic
Substances
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CRISPR-Associated Proteins
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RNA, Guide, CRISPR-Cas Systems
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RNA, Transfer
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RNA Polymerase II
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RNA Polymerase III