Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency
| Autor: | Gengxiang Jia, Hongming Ma, Ying Dang, Haoquan Wu, Premlata Shankar, Chunting Ye, Edgar Anaya, Jennie Choi |
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| Rok vydání: | 2015 |
| Předmět: |
Guanine
CRISPR-Associated Proteins Computational biology Biology Cell Line Gene Knockout Techniques Jurkat Cells 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Transcription (biology) Deoxyribonuclease I Humans CRISPR Gene knockout 030304 developmental biology Trans-activating crRNA Genetics 0303 health sciences Research RNA chemistry Mutation CRISPR-Cas Systems Gene Deletion 030217 neurology & neurosurgery Cytosine |
| Zdroj: | Genome Biology |
| ISSN: | 1474-760X |
| DOI: | 10.1186/s13059-015-0846-3 |
| Popis: | 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. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0846-3) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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