Enhancement of single guide RNA transcription for efficient CRISPR/Cas-based genomic engineering.
Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein. A widely used gRNA is an RNA polymerase III (pol III)-driven single gRNA (sgRNA), which is produced by artificial fusion of CRISPR RNA (crRNA) and trans-activation crRNA (tracrRNA). However, we identified a TTTT stretch, known as a termination signal of RNA pol III, in the scaffold region of the sgRNA. Here, we revealed that sgRNA carrying a TTTT stretch reduces the efficiency of sgRNA transcription due to premature transcriptional termination, and decreases the efficiency of genome editing. Unexpectedly, it was also shown that the premature terminated sgRNA may have an adverse effect of inducing RNA interference. Such disadvantageous effects were avoided by substituting one base in the TTTT stretch.
a Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba-ken 277-8651, Japan.
Kumiko
K
Ui-Tei
b Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba-ken 277-8561, Japan.
Shohei
S
Maruyama
c Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Yuko
Y
Nakano
D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004247:DNA / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D016678:Genome / Q000235:genetics*; D023281:Genomics / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D012313:RNA / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*
D000094704:RNA, Guide, CRISPR-Cas Systems; D012313:RNA; D004247:DNA; D012321:DNA-Directed RNA Polymerases
ARN polymérase III; CRISPR; Cas9; RNA interference; RNA polymerase III; interférence à l’ARN; sgRNA