We are analyzing https://link.springer.com/article/10.1186/s13059-015-0846-3.

Title:
Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency | Genome Biology
Description:
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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Keywords {🔍}

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         headline:Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency
         description: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. 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. 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.
         datePublished:2015-12-15T00:00:00Z
         dateModified:2015-12-15T00:00:00Z
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            Cluster Regularly Interspaced Short Palindromic Repeat
            Continuous Sequence
            CCR5 Gene
            Green Fluorescent Protein Fusion Protein
            Animal Genetics and Genomics
            Human Genetics
            Plant Genetics and Genomics
            Microbial Genetics and Genomics
            Bioinformatics
            Evolutionary Biology
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      headline:Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency
      description: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. 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. 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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         Cluster Regularly Interspaced Short Palindromic Repeat
         Continuous Sequence
         CCR5 Gene
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         Animal Genetics and Genomics
         Human Genetics
         Plant Genetics and Genomics
         Microbial Genetics and Genomics
         Bioinformatics
         Evolutionary Biology
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      name:Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, USA
      name:Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, USA
      name:Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, USA
      name:Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, USA
      name:Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, USA
      name:Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, USA
      name:Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, USA
      name:Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, USA

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