Rescue of high-specificity Cas9 variants using sgRNAs with matched 5’ nucleotides
Abstract We report that engineered Cas9 variants with improved specificity—eCas9-1.1 and Cas9-HF1—are often poorly active in human cells, when complexed with single guide RNAs (sgRNAs) with a mismatch at the 5’ terminus, relative to target DNA sequences. Because the nucleotide at the 5’ end of sgRNA...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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BMC
2017-11-01
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| Series: | Genome Biology |
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| Online Access: | http://link.springer.com/article/10.1186/s13059-017-1355-3 |
| _version_ | 1818060247904813056 |
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| author | Sojung Kim Taegeun Bae Jaewoong Hwang Jin-Soo Kim |
| author_facet | Sojung Kim Taegeun Bae Jaewoong Hwang Jin-Soo Kim |
| author_sort | Sojung Kim |
| collection | DOAJ |
| description | Abstract We report that engineered Cas9 variants with improved specificity—eCas9-1.1 and Cas9-HF1—are often poorly active in human cells, when complexed with single guide RNAs (sgRNAs) with a mismatch at the 5’ terminus, relative to target DNA sequences. Because the nucleotide at the 5’ end of sgRNAs, expressed under the control of the commonly-used U6 promoter, is fixed to a guanine, these attenuated Cas9 variants are not useful at many target sites. By using sgRNAs with matched 5’ nucleotides, produced by linking them to a self-cleaving ribozyme, the editing activity of Cas9 variants can be rescued without sacrificing high specificity. |
| first_indexed | 2024-12-10T13:29:24Z |
| format | Article |
| id | doaj.art-a538a35628144034aace727b8f5df66c |
| institution | Directory Open Access Journal |
| issn | 1474-760X |
| language | English |
| last_indexed | 2024-12-10T13:29:24Z |
| publishDate | 2017-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Genome Biology |
| spelling | doaj.art-a538a35628144034aace727b8f5df66c2022-12-22T01:47:02ZengBMCGenome Biology1474-760X2017-11-011811610.1186/s13059-017-1355-3Rescue of high-specificity Cas9 variants using sgRNAs with matched 5’ nucleotidesSojung Kim0Taegeun Bae1Jaewoong Hwang2Jin-Soo Kim3Center for Genome Engineering, Institute for Basic ScienceCenter for Genome Engineering, Institute for Basic ScienceDepartment of Chemistry, Seoul National UniversityCenter for Genome Engineering, Institute for Basic ScienceAbstract We report that engineered Cas9 variants with improved specificity—eCas9-1.1 and Cas9-HF1—are often poorly active in human cells, when complexed with single guide RNAs (sgRNAs) with a mismatch at the 5’ terminus, relative to target DNA sequences. Because the nucleotide at the 5’ end of sgRNAs, expressed under the control of the commonly-used U6 promoter, is fixed to a guanine, these attenuated Cas9 variants are not useful at many target sites. By using sgRNAs with matched 5’ nucleotides, produced by linking them to a self-cleaving ribozyme, the editing activity of Cas9 variants can be rescued without sacrificing high specificity.http://link.springer.com/article/10.1186/s13059-017-1355-3CRISPR-CasOff-target effectEngineered Cas9 variantsHammerhead ribozyme-linked sgRNA |
| spellingShingle | Sojung Kim Taegeun Bae Jaewoong Hwang Jin-Soo Kim Rescue of high-specificity Cas9 variants using sgRNAs with matched 5’ nucleotides Genome Biology CRISPR-Cas Off-target effect Engineered Cas9 variants Hammerhead ribozyme-linked sgRNA |
| title | Rescue of high-specificity Cas9 variants using sgRNAs with matched 5’ nucleotides |
| title_full | Rescue of high-specificity Cas9 variants using sgRNAs with matched 5’ nucleotides |
| title_fullStr | Rescue of high-specificity Cas9 variants using sgRNAs with matched 5’ nucleotides |
| title_full_unstemmed | Rescue of high-specificity Cas9 variants using sgRNAs with matched 5’ nucleotides |
| title_short | Rescue of high-specificity Cas9 variants using sgRNAs with matched 5’ nucleotides |
| title_sort | rescue of high specificity cas9 variants using sgrnas with matched 5 nucleotides |
| topic | CRISPR-Cas Off-target effect Engineered Cas9 variants Hammerhead ribozyme-linked sgRNA |
| url | http://link.springer.com/article/10.1186/s13059-017-1355-3 |
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