Protein Engineering Strategies to Expand CRISPR-Cas9 Applications

Author(s)

Ribeiro, Lucas F. ; Ribeiro, Liliane F. C. ; Barreto, Matheus Q. ; Ward, Richard J.

• Document type: Journal article
• Source: INTERNATIONAL JOURNAL OF GENOMICS; v. 2018, p. 12-pg., 2018-01-01.

Abstract

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. (AU)

• FAPESP's process: 16/18827-7 - Integrating protein engineering and synthetic biology to develop new high-throughput selection systems
• Grantee: Lucas Ferreira Ribeiro
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 16/20358-5 - Systems biology analysis of the role of protein kinase A (PKA), in the fungus Trichoderma reesei by phosphoproteomic
• Grantee: Liliane Fraga Costa Ribeiro
• Support Opportunities: Scholarships in Brazil - Post-Doctoral