Abstract
BackgroundSingle-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.ResultsHere, 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.ConclusionsBy 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 materialThe online version of this article (doi:10.1186/s13059-015-0846-3) contains supplementary material, which is available to authorized users.
Highlights
Single-guide RNA is one of the two key components of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system
The current most commonly used Single-guide RNA (sgRNA) design has the duplex shortened by 10 bp compared with the native CRISPR RNA (crRNA)–tracrRNA duplex (Fig. 1a), which does not seem to reduce its functionality in vitro [6]
We extended the duplex in two sgRNAs targeting the CCR5 gene, as shown in Fig. 1a, and determined the knockout efficiency of these mutants in TZM
Summary
Single-guide RNA (sgRNA) is one of the two key components of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas 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 could potentially reduce transcription efficiency. The clustered regularly interspaced short palindromic repeats (CRISPR) system has recently been developed into a powerful genome-editing technology [1,2,3,4,5,6] This system is composed of two components: the nuclease Cas and the guide RNA. We systematically investigated the effect of changing these two elements on knockout efficiency and found that, overall, extending the duplex and mutating the continuous sequence of Ts significantly improved knockout efficiency
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