The DOI matches its Crossref record; 11 of the 11 references it cites were found in a registry or an index.
Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency
Open accessAbstract
BACKGROUND: Single-guide RNA (sgRNA) is one of the two key components of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system. The current commonly used sgRNA structure has a shortened duplex compared with the native bacterial CRISPR RNA (crRNA)-transactivating crRNA (tracrRNA) duplex and contains a continuous sequence of thymines, which is the pause signal for RNA polymerase III and thus could potentially reduce transcription efficiency. RESULTS: Here, we systematically investigate the effect of these two elements on knockout efficiency and showed that modifying the sgRNA structure by extending the duplex length and mutating the fourth thymine of the continuous sequence of thymines to cytosine or guanine significantly, and sometimes dramatically, improves knockout efficiency in cells. In addition, the optimized sgRNA structure also significantly increases the efficiency of more challenging genome-editing procedures, such as gene deletion, which is important for inducing a loss of function in non-coding genes. CONCLUSIONS: By a systematic investigation of sgRNA structure we find that extending the duplex by approximately 5 bp combined with mutating the continuous sequence of thymines at position 4 to cytosine or guanine significantly increases gene knockout efficiency in CRISPR-Cas9-based genome editing experiments.
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Cites
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Cited by
Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities
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The CRISPR-Cas toolbox and gene editing technologies
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Combinatorial CRISPR–Cas9 screens for de novo mapping of genetic interactions
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Metadata
- Authors
- Dang, Y., Jia, G., Choi, J., Ma, H., Anaya, E., Ye, C., Shankar, P., & Wu, H.
- Venue
- Genome Biology
- Published
- 2015-12
- Volume
- 16
- Issue
- 1
- Pages
- 280-280
- ISSN
- 1465-6906 (ISSN-L)
- 1474-760X
- OpenAlex
- W2203807603
- Cited by
- 364
- References
- 11
- FWCI
- 9.26
- Type
- journal article
- Language
- en
- Open access
- gold
- Licence
- creativecommons.org/licenses/by/4.0/ (tdm)
- creativecommons.org/licenses/by/4.0/ (vor, 14 d)
- Funders
- National Institute of Allergy and Infectious Diseases (1R56AI114357)
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- Dang, Y., Jia, G., Choi, J., Ma, H., Anaya, E., Ye, C., Shankar, P., & Wu, H. (2015). Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency. Genome Biology. https://doi.org/10.1186/s13059-015-0846-3
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