According to Greg Davis, Ph.D., principal R&D scientist, Sigma-Aldrich Biotechnology, key ZFN enhancements in recent years are cleavage domains, termed enhanced high-fidelity FokI domains. Because these domains work only as heterodimers, they enhance specificity by blocking an individual ZFN from homodimerizing with itself on DNA and cleaving potential off-target sites.
Whereas ZFNs require two binding events to create the double-strand DNA break, the CRISPR technology initially required just one binding event. To improve the specificity of the CRISPR/Cas9 technology, paired nickases seemed like a tractable approach.
If two CRISPRs bind close together and both nick an opposite strand of DNA, a double-strand break will occur. At Sigma, Cas9 mutations were undertaken to create nickases to cleave only one strand of DNA. Close nicks result in a double-strand break. Nickases allow more permissive spacing than the use of FokI, providing increased flexibility for practical research applications, such as disease SNP modeling.
Sigma has a large lentivirus vector library, used currently for shRNA libraries, and is applying it to CRISPR. shRNA screening is limited to the exome of the genome, the protein-coding genes or roughly 1% of the genome, whereas CRISPR targets the entire chromosome. A good delivery tool, lentivirus is applicable to both arrayed and pooled high-throughput screening applications.
RNA interference (RNAi) represses activity, but for activation and epigenetic studies, modified ZFNs, TALENs, and CRISPRs may be applicable. A natural extension to the CRISPR platform is to inactivate the nuclease activity on the Cas9 protein, turning the CRISPR into a DNA-binding protein. Then activators, such as VP16 and VP64. and repressors, such as the KRAB domain, can be fused to dead Cas9. This allows enzymatic activity to be localized to a specific part of the chromosome, permitting the study of genetic regulation at specific loci.
Sigma has formed a partnership with Cleveland Clinic’s Molecular Screening Core to develop a CRISPR core. This new core is part of the Case Comprehensive Cancer Center at Case Western Reserve University and is open for researchers at Cleveland Clinic and other biomedical research centers in the Cleveland area.
One of core’s first users, Paul Tesar, Ph.D., associate professor, department of genetics and genome sciences, Case Western Reserve University School of Medicine, uses gene-editing technology to study neurogenic and neurodevelopment disorders. Dr. Tesar’s group focuses on areas of the brain that impact oligodendrocytes, which make myelin.
Specific mutations cause oligodendrocytes in the brain to produce myelin less effectively. The result: leukodystrophies, a class of pediatric congenital disorders. To better understand these diseases, the laboratory uses genome editing and aspires to create corrected oligodendrocytes from pluripotent stem cells. Once modified, the oligodendrocytes would help correct the disease in these patients.
“Gene-editing technology continually changes and can be overwhelming to a new user,” commented Dr. Tesar. “The new CRISPR core provides a rapid and effective way to access the technology regardless of level of expertise, and builds a community of researchers. Protocols, vectors, support, and technical expertise are in place to help you get exactly what you need for your particular experiment.”