Synthetic switch to minimize CRISPR off-target effects by self-restricting Cas9 transcription and translation
Abstract CRISPR/Cas9 is a powerful genome editing system but uncontrolled Cas9 nuclease expression triggers off-target effects and even in vivo immune responses. Inspired by synthetic biology, here we built a synthetic switch that self-regulates Cas9 expression not only in the transcription step by...
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| Published in | Nucleic acids research Vol. 47; no. 3; p. e13 |
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| Main Authors | , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
England
Oxford University Press
20.02.2019
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| Subjects | |
| Online Access | Get full text |
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| Abstract | Abstract
CRISPR/Cas9 is a powerful genome editing system but uncontrolled Cas9 nuclease expression triggers off-target effects and even in vivo immune responses. Inspired by synthetic biology, here we built a synthetic switch that self-regulates Cas9 expression not only in the transcription step by guide RNA-aided self-cleavage of cas9 gene, but also in the translation step by L7Ae:K-turn repression system. We showed that the synthetic switch enabled simultaneous transcriptional and translational repression, hence stringently attenuating the Cas9 expression. The restricted Cas9 expression induced high efficiency on-target indel mutation while minimizing the off-target effects. Furthermore, we unveiled the correlation between Cas9 expression kinetics and on-target/off-target mutagenesis. The synthetic switch conferred detectable Cas9 expression and concomitant high frequency on-target mutagenesis at as early as 6 h, and restricted the Cas9 expression and off-target effects to minimal levels through 72 h. The synthetic switch is compact enough to be incorporated into viral vectors for self-regulation of Cas9 expression, thereby providing a novel ‘hit and run’ strategy for in vivo genome editing. |
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| AbstractList | CRISPR/Cas9 is a powerful genome editing system but uncontrolled Cas9 nuclease expression triggers off-target effects and even
in vivo
immune responses. Inspired by synthetic biology, here we built a synthetic switch that self-regulates Cas9 expression not only in the transcription step by guide RNA-aided self-cleavage of
cas9
gene, but also in the translation step by L7Ae:K-turn repression system. We showed that the synthetic switch enabled simultaneous transcriptional and translational repression, hence stringently attenuating the Cas9 expression. The restricted Cas9 expression induced high efficiency on-target indel mutation while minimizing the off-target effects. Furthermore, we unveiled the correlation between Cas9 expression kinetics and on-target/off-target mutagenesis. The synthetic switch conferred detectable Cas9 expression and concomitant high frequency on-target mutagenesis at as early as 6 h, and restricted the Cas9 expression and off-target effects to minimal levels through 72 h. The synthetic switch is compact enough to be incorporated into viral vectors for self-regulation of Cas9 expression, thereby providing a novel ‘hit and run’ strategy for
in vivo
genome editing. CRISPR/Cas9 is a powerful genome editing system but uncontrolled Cas9 nuclease expression triggers off-target effects and even in vivo immune responses. Inspired by synthetic biology, here we built a synthetic switch that self-regulates Cas9 expression not only in the transcription step by guide RNA-aided self-cleavage of cas9 gene, but also in the translation step by L7Ae:K-turn repression system. We showed that the synthetic switch enabled simultaneous transcriptional and translational repression, hence stringently attenuating the Cas9 expression. The restricted Cas9 expression induced high efficiency on-target indel mutation while minimizing the off-target effects. Furthermore, we unveiled the correlation between Cas9 expression kinetics and on-target/off-target mutagenesis. The synthetic switch conferred detectable Cas9 expression and concomitant high frequency on-target mutagenesis at as early as 6 h, and restricted the Cas9 expression and off-target effects to minimal levels through 72 h. The synthetic switch is compact enough to be incorporated into viral vectors for self-regulation of Cas9 expression, thereby providing a novel 'hit and run' strategy for in vivo genome editing.CRISPR/Cas9 is a powerful genome editing system but uncontrolled Cas9 nuclease expression triggers off-target effects and even in vivo immune responses. Inspired by synthetic biology, here we built a synthetic switch that self-regulates Cas9 expression not only in the transcription step by guide RNA-aided self-cleavage of cas9 gene, but also in the translation step by L7Ae:K-turn repression system. We showed that the synthetic switch enabled simultaneous transcriptional and translational repression, hence stringently attenuating the Cas9 expression. The restricted Cas9 expression induced high efficiency on-target indel mutation while minimizing the off-target effects. Furthermore, we unveiled the correlation between Cas9 expression kinetics and on-target/off-target mutagenesis. The synthetic switch conferred detectable Cas9 expression and concomitant high frequency on-target mutagenesis at as early as 6 h, and restricted the Cas9 expression and off-target effects to minimal levels through 72 h. The synthetic switch is compact enough to be incorporated into viral vectors for self-regulation of Cas9 expression, thereby providing a novel 'hit and run' strategy for in vivo genome editing. CRISPR/Cas9 is a powerful genome editing system but uncontrolled Cas9 nuclease expression triggers off-target effects and even in vivo immune responses. Inspired by synthetic biology, here we built a synthetic switch that self-regulates Cas9 expression not only in the transcription step by guide RNA-aided self-cleavage of cas9 gene, but also in the translation step by L7Ae:K-turn repression system. We showed that the synthetic switch enabled simultaneous transcriptional and translational repression, hence stringently attenuating the Cas9 expression. The restricted Cas9 expression induced high efficiency on-target indel mutation while minimizing the off-target effects. Furthermore, we unveiled the correlation between Cas9 expression kinetics and on-target/off-target mutagenesis. The synthetic switch conferred detectable Cas9 expression and concomitant high frequency on-target mutagenesis at as early as 6 h, and restricted the Cas9 expression and off-target effects to minimal levels through 72 h. The synthetic switch is compact enough to be incorporated into viral vectors for self-regulation of Cas9 expression, thereby providing a novel 'hit and run' strategy for in vivo genome editing. Abstract CRISPR/Cas9 is a powerful genome editing system but uncontrolled Cas9 nuclease expression triggers off-target effects and even in vivo immune responses. Inspired by synthetic biology, here we built a synthetic switch that self-regulates Cas9 expression not only in the transcription step by guide RNA-aided self-cleavage of cas9 gene, but also in the translation step by L7Ae:K-turn repression system. We showed that the synthetic switch enabled simultaneous transcriptional and translational repression, hence stringently attenuating the Cas9 expression. The restricted Cas9 expression induced high efficiency on-target indel mutation while minimizing the off-target effects. Furthermore, we unveiled the correlation between Cas9 expression kinetics and on-target/off-target mutagenesis. The synthetic switch conferred detectable Cas9 expression and concomitant high frequency on-target mutagenesis at as early as 6 h, and restricted the Cas9 expression and off-target effects to minimal levels through 72 h. The synthetic switch is compact enough to be incorporated into viral vectors for self-regulation of Cas9 expression, thereby providing a novel ‘hit and run’ strategy for in vivo genome editing. |
| Author | Lin, Mei-Wei Hwu, Jih-Ru Hsu, Mu-Nung Shen, Chih-Che Tu, Yi Hu, Yu-Chen Chang, Chin-Wei |
| AuthorAffiliation | 5 Department of Life Science, National Taiwan University, Taipei, Taiwan 3 Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan 2 Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan 1 Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan 4 Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu, Taiwan |
| AuthorAffiliation_xml | – name: 1 Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan – name: 3 Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan – name: 4 Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu, Taiwan – name: 5 Department of Life Science, National Taiwan University, Taipei, Taiwan – name: 2 Biomedical Technology and Device Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan |
| Author_xml | – sequence: 1 givenname: Chih-Che surname: Shen fullname: Shen, Chih-Che organization: Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan – sequence: 2 givenname: Mu-Nung surname: Hsu fullname: Hsu, Mu-Nung organization: Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan – sequence: 3 givenname: Chin-Wei surname: Chang fullname: Chang, Chin-Wei organization: Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan – sequence: 4 givenname: Mei-Wei surname: Lin fullname: Lin, Mei-Wei organization: Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan – sequence: 5 givenname: Jih-Ru surname: Hwu fullname: Hwu, Jih-Ru organization: Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan – sequence: 6 givenname: Yi surname: Tu fullname: Tu, Yi organization: Department of Life Science, National Taiwan University, Taipei, Taiwan – sequence: 7 givenname: Yu-Chen orcidid: 0000-0002-9997-4467 surname: Hu fullname: Hu, Yu-Chen email: ychu@mx.nthu.edu.tw organization: Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/30462300$$D View this record in MEDLINE/PubMed |
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CRISPR/Cas9 is a powerful genome editing system but uncontrolled Cas9 nuclease expression triggers off-target effects and even in vivo immune... CRISPR/Cas9 is a powerful genome editing system but uncontrolled Cas9 nuclease expression triggers off-target effects and even in vivo immune responses.... CRISPR/Cas9 is a powerful genome editing system but uncontrolled Cas9 nuclease expression triggers off-target effects and even in vivo immune responses.... |
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| SubjectTerms | Cell Line CRISPR-Associated Protein 9 - biosynthesis CRISPR-Associated Protein 9 - genetics CRISPR-Cas Systems Gene Editing Gene Expression Regulation Humans Kinetics Methods Online Mutagenesis Mutation Protein Biosynthesis Transcription, Genetic |
| Title | Synthetic switch to minimize CRISPR off-target effects by self-restricting Cas9 transcription and translation |
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