Protein Engineering Strategies to Expand CRISPR-Cas9 Applications
The development of precise and modulated methods for customized manipulation of DNA is an important objective for the study and engineering of biological processes and is essential for the optimization of gene therapy, metabolic flux, and synthetic gene networks. The clustered regularly interspaced...
Saved in:
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2018-01-01
|
| Series: | International Journal of Genomics |
| Online Access: | http://dx.doi.org/10.1155/2018/1652567 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850166966221799424 |
|---|---|
| author | Lucas F. Ribeiro Liliane F. C. Ribeiro Matheus Q. Barreto Richard J. Ward |
| author_facet | Lucas F. Ribeiro Liliane F. C. Ribeiro Matheus Q. Barreto Richard J. Ward |
| author_sort | Lucas F. Ribeiro |
| collection | DOAJ |
| description | The development of precise and modulated methods for customized manipulation of DNA is an important objective for the study and engineering of biological processes and is essential for the optimization of gene therapy, metabolic flux, and synthetic gene networks. The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 is an RNA-guided site-specific DNA-binding complex that can be reprogrammed to specifically interact with a desired DNA sequence target. CRISPR-Cas9 has been used in a wide variety of applications ranging from basic science to the clinic, such as gene therapy, gene regulation, modifying epigenomes, and imaging chromosomes. Although Cas9 has been successfully used as a precise tool in all these applications, some limitations have also been reported, for instance (i) a strict dependence on a protospacer-adjacent motif (PAM) sequence, (ii) aberrant off-target activity, (iii) the large size of Cas9 is problematic for CRISPR delivery, and (iv) lack of modulation of protein binding and endonuclease activity, which is crucial for precise spatiotemporal control of gene expression or genome editing. These obstacles hinder the use of CRISPR for disease treatment and in wider biotechnological applications. Protein-engineering approaches offer solutions to overcome the limitations of Cas9 and generate robust and efficient tools for customized DNA manipulation. Here, recent protein-engineering approaches for expanding the versatility of the Streptococcus pyogenes Cas9 (SpCas9) is reviewed, with an emphasis on studies that improve or develop novel protein functions through domain fusion or splitting, rational design, and directed evolution. |
| format | Article |
| id | doaj-art-e4b59fcaacfd4bbe91514231dcda8a97 |
| institution | OA Journals |
| issn | 2314-436X 2314-4378 |
| language | English |
| publishDate | 2018-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | International Journal of Genomics |
| spelling | doaj-art-e4b59fcaacfd4bbe91514231dcda8a972025-08-20T02:21:19ZengWileyInternational Journal of Genomics2314-436X2314-43782018-01-01201810.1155/2018/16525671652567Protein Engineering Strategies to Expand CRISPR-Cas9 ApplicationsLucas F. Ribeiro0Liliane F. C. Ribeiro1Matheus Q. Barreto2Richard J. Ward3Department of Biology, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, University of São Paulo, São Paulo, SP, BrazilDepartment of Biochemistry and Immunology, Faculdade de Medicina de Ribeirão Preto, University of São Paulo, São Paulo, SP, BrazilDepartment of Biochemistry and Immunology, Faculdade de Medicina de Ribeirão Preto, University of São Paulo, São Paulo, SP, BrazilDepartment of Chemistry, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, University of São Paulo, São Paulo, SP, BrazilThe development of precise and modulated methods for customized manipulation of DNA is an important objective for the study and engineering of biological processes and is essential for the optimization of gene therapy, metabolic flux, and synthetic gene networks. The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 is an RNA-guided site-specific DNA-binding complex that can be reprogrammed to specifically interact with a desired DNA sequence target. CRISPR-Cas9 has been used in a wide variety of applications ranging from basic science to the clinic, such as gene therapy, gene regulation, modifying epigenomes, and imaging chromosomes. Although Cas9 has been successfully used as a precise tool in all these applications, some limitations have also been reported, for instance (i) a strict dependence on a protospacer-adjacent motif (PAM) sequence, (ii) aberrant off-target activity, (iii) the large size of Cas9 is problematic for CRISPR delivery, and (iv) lack of modulation of protein binding and endonuclease activity, which is crucial for precise spatiotemporal control of gene expression or genome editing. These obstacles hinder the use of CRISPR for disease treatment and in wider biotechnological applications. Protein-engineering approaches offer solutions to overcome the limitations of Cas9 and generate robust and efficient tools for customized DNA manipulation. Here, recent protein-engineering approaches for expanding the versatility of the Streptococcus pyogenes Cas9 (SpCas9) is reviewed, with an emphasis on studies that improve or develop novel protein functions through domain fusion or splitting, rational design, and directed evolution.http://dx.doi.org/10.1155/2018/1652567 |
| spellingShingle | Lucas F. Ribeiro Liliane F. C. Ribeiro Matheus Q. Barreto Richard J. Ward Protein Engineering Strategies to Expand CRISPR-Cas9 Applications International Journal of Genomics |
| title | Protein Engineering Strategies to Expand CRISPR-Cas9 Applications |
| title_full | Protein Engineering Strategies to Expand CRISPR-Cas9 Applications |
| title_fullStr | Protein Engineering Strategies to Expand CRISPR-Cas9 Applications |
| title_full_unstemmed | Protein Engineering Strategies to Expand CRISPR-Cas9 Applications |
| title_short | Protein Engineering Strategies to Expand CRISPR-Cas9 Applications |
| title_sort | protein engineering strategies to expand crispr cas9 applications |
| url | http://dx.doi.org/10.1155/2018/1652567 |
| work_keys_str_mv | AT lucasfribeiro proteinengineeringstrategiestoexpandcrisprcas9applications AT lilianefcribeiro proteinengineeringstrategiestoexpandcrisprcas9applications AT matheusqbarreto proteinengineeringstrategiestoexpandcrisprcas9applications AT richardjward proteinengineeringstrategiestoexpandcrisprcas9applications |