Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian Cells
CRISPR-Cas9 is quickly revolutionizing the way we approach gene therapy. CRISPR-Cas9 is a complexed, two-component system using a short guide RNA (gRNA) sequence to direct the Cas9 endonuclease to the target site. Modifying the gRNA independent of the Cas9 protein confers ease and flexibility to imp...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2021-01-01
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| Series: | Frontiers in Genome Editing |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fgeed.2020.617910/full |
| _version_ | 1818339205851381760 |
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| author | Daniel Allen Michael Rosenberg Ayal Hendel |
| author_facet | Daniel Allen Michael Rosenberg Ayal Hendel |
| author_sort | Daniel Allen |
| collection | DOAJ |
| description | CRISPR-Cas9 is quickly revolutionizing the way we approach gene therapy. CRISPR-Cas9 is a complexed, two-component system using a short guide RNA (gRNA) sequence to direct the Cas9 endonuclease to the target site. Modifying the gRNA independent of the Cas9 protein confers ease and flexibility to improve the CRISPR-Cas9 system as a genome-editing tool. gRNAs have been engineered to improve the CRISPR system's overall stability, specificity, safety, and versatility. gRNAs have been modified to increase their stability to guard against nuclease degradation, thereby enhancing their efficiency. Additionally, guide specificity has been improved by limiting off-target editing. Synthetic gRNA has been shown to ameliorate inflammatory signaling caused by the CRISPR system, thereby limiting immunogenicity and toxicity in edited mammalian cells. Furthermore, through conjugation with exogenous donor DNA, engineered gRNAs have been shown to improve homology-directed repair (HDR) efficiency by ensuring donor proximity to the edited site. Lastly, synthetic gRNAs attached to fluorescent labels have been developed to enable highly specific nuclear staining and imaging, enabling mechanistic studies of chromosomal dynamics and genomic mapping. Continued work on chemical modification and optimization of synthetic gRNAs will undoubtedly lead to clinical and therapeutic benefits and, ultimately, routinely performed CRISPR-based therapies. |
| first_indexed | 2024-12-13T15:23:19Z |
| format | Article |
| id | doaj.art-153c5e30a01f41efb8e97ce6ac159ebb |
| institution | Directory Open Access Journal |
| issn | 2673-3439 |
| language | English |
| last_indexed | 2024-12-13T15:23:19Z |
| publishDate | 2021-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Genome Editing |
| spelling | doaj.art-153c5e30a01f41efb8e97ce6ac159ebb2022-12-21T23:40:28ZengFrontiers Media S.A.Frontiers in Genome Editing2673-34392021-01-01210.3389/fgeed.2020.617910617910Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian CellsDaniel AllenMichael RosenbergAyal HendelCRISPR-Cas9 is quickly revolutionizing the way we approach gene therapy. CRISPR-Cas9 is a complexed, two-component system using a short guide RNA (gRNA) sequence to direct the Cas9 endonuclease to the target site. Modifying the gRNA independent of the Cas9 protein confers ease and flexibility to improve the CRISPR-Cas9 system as a genome-editing tool. gRNAs have been engineered to improve the CRISPR system's overall stability, specificity, safety, and versatility. gRNAs have been modified to increase their stability to guard against nuclease degradation, thereby enhancing their efficiency. Additionally, guide specificity has been improved by limiting off-target editing. Synthetic gRNA has been shown to ameliorate inflammatory signaling caused by the CRISPR system, thereby limiting immunogenicity and toxicity in edited mammalian cells. Furthermore, through conjugation with exogenous donor DNA, engineered gRNAs have been shown to improve homology-directed repair (HDR) efficiency by ensuring donor proximity to the edited site. Lastly, synthetic gRNAs attached to fluorescent labels have been developed to enable highly specific nuclear staining and imaging, enabling mechanistic studies of chromosomal dynamics and genomic mapping. Continued work on chemical modification and optimization of synthetic gRNAs will undoubtedly lead to clinical and therapeutic benefits and, ultimately, routinely performed CRISPR-based therapies.https://www.frontiersin.org/articles/10.3389/fgeed.2020.617910/fullCRISPR-Cas9engineered nucleasegRNAchemical modificationsgenome editinggene therapy |
| spellingShingle | Daniel Allen Michael Rosenberg Ayal Hendel Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian Cells Frontiers in Genome Editing CRISPR-Cas9 engineered nuclease gRNA chemical modifications genome editing gene therapy |
| title | Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian Cells |
| title_full | Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian Cells |
| title_fullStr | Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian Cells |
| title_full_unstemmed | Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian Cells |
| title_short | Using Synthetically Engineered Guide RNAs to Enhance CRISPR Genome Editing Systems in Mammalian Cells |
| title_sort | using synthetically engineered guide rnas to enhance crispr genome editing systems in mammalian cells |
| topic | CRISPR-Cas9 engineered nuclease gRNA chemical modifications genome editing gene therapy |
| url | https://www.frontiersin.org/articles/10.3389/fgeed.2020.617910/full |
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