Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs.
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.
Cell Biology Division, MRC Laboratory of Molecular Biology, Cambridge, UK.
Fillip
F
Port
Cell Biology Division, MRC Laboratory of Molecular Biology, Cambridge, UK.
Simon L
0000-0001-9491-4548
SL
Bullock
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics; D064130:CRISPR-Associated Proteins / Q000235:genetics*; D064113:CRISPR-Cas Systems / Q000235:genetics; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004330:Drosophila / Q000201:enzymology / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017393:RNA Editing / Q000235:genetics; D012319:RNA Polymerase II / Q000235:genetics; D012320:RNA Polymerase III / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics*; D014158:Transcription, Genetic
D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D012343:RNA, Transfer; D012319:RNA Polymerase II; D012320:RNA Polymerase III