Studying mutants that have acquired changes or deletions in DNA sequences leading to alternate phenotypes is a time-honored research strategy to define spatial and temporal functional properties of the genes. The currently ongoing genetic studies, as well as the data accrued over the past half century will reveal a collection of novel and known mutations in new and previously identified genes responsible for various ocular diseases such as Retinitis Pigmentosa, Stargardt disease, Keratoconus, etc based on inheritance patterns. To this end the clinical phenotype is correlated to the genotypes and patients are counselled in the Gen-Eye clinic.
Translating the knowledge derived from patient genetic studies
Thereafter, it is important to determine how mutations in genes and subsequent alterations in their encoded proteins function in the patient. In recent years, new genome editing tools such as zinc-finger nucleases, TALENs and the more recent CRISPR-cas9 systems have emerged as high efficiency methods to change nucleotide sequences as per the user's choice. This allows for creating paired cell lines with specific mutations that can mimic the patient discoveries and therefore studies biochemically. In addition, the development of primary cell cultures and pluripotent stem cells from human patients/donors has helped study gene functions in vitro with greater efficiency. We take advantage of these tools in the current study to further understand functional roles of the mutations discovered in Indian patients. These strategies can be harnessed together to develop patient specific therapeutic strategies for treatment of genetic diseases.
Investigating functional genetics using genome editing tool CRISPR-Cas9 system
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Type II system has been modified for genome engineering after adapating it from the bacterial immune system. CRISPR was originally employed to “knock-out” target genes in various cell types and organisms, but modifications to the Cas9 enzyme have extended the application of CRISPR to selectively activate or repress target genes, purify specific regions of DNA, and even image DNA in live cells using fluorescence microscopy. The CRISPR/Cas9 system requires a custom sgRNA sequence (user-defined ∼20 nucleotide “spacer” or “targeting” sequence to direct Cas9 nuclease activity plus “scaffold” sequence necessary for Cas9-binding) and Cas9 (non-specific CRISPR-associated Endonuclease) nuclease-recruiting sequence. The genomic target of Cas9 can be changed by simply changing the targeting sequence present in the gRNA and this makes the procedure very convenient for specific or large scale genomic modifications. Due to its simplicity and adaptability, CRISPR/Cas9 genome editing has rapidly become one of the most popular approaches for in vitro and germ-line genome editing including targeted correction of specific mutations. We are currently using this strategy to establish in vitro disease models for elucidating molecular mechanisms and testing therapeutic strategies.