The CRISPR-Cas9 genome-engineering tool is a powerful opportunity for researchers to study individual gene function. CRISPR-Cas9, abbreviated for Clustered Regularly Interspaced Short Palindromic Repeats, is a bacterial defense system that can be reprogrammed to target specific areas of DNA followed by precise editing. Essentially, CRISPR sequences are transcribed into short RNA sequences that will match the desired DNA sequence of interest. From here, the DNA is bound and cut, turning off its function. While this recent discovery opened up doors for gene-targeted therapies, the mechanisms in which DNA cutting was achieved are somewhat debated. Simply put, when DNA is damaged, it sets out to repair itself, resulting in randomized nucleic acid insertions that are not needed.
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CRISPR-Cas9 Antibody (7A9-3A3) [NBP2-36440] - ICC/IF analysis of Crispr-Cas9 transfected HEK293 cells using CRISPR-Cas9 antibody (clone 7A9-3A3). Red staining represents CRISPR-Cas9 positivity while DAPI stained nuclei are visible in blue color.
A group out of Harvard recently published a more sophisticated version of CRISPR in Nature Magazine that uses a less aggressive method of DNA interruption, especially when it comes to point mutations. Their development, referred to as “base editing” allows for direct conversion of one DNA base pair to another without double stranded DNA breaks. Specifically, the direct conversion between cytidine to uridine is mediated by the new system, which in turn activates the substitution. This is especially helpful where entire gene deletions are not required to rescue the effects of disease pathology, but rather specific point mutations.
Additionally, genome scale screening tools are being developed and utilized. These tools are aimed at increasing the efficiency of cell knockouts in bulk populations, possibly even using homology directed repair (HDR) to repair double strand breaks using a targeted marker or resistance gene. There are also initiatives addressing RNA inactivation challenges, where inactivating gene activity at a single-copy integration of the shRNA expression cassette is difficult. At this time, databases containing CRISPR-Cas9 screenings are including independent reagents for each gene, which helps to avoid false negatives.
Lastly, CRISPR-Cas9 has graduated from in vitro cell studies to in vivo studies, where it may rapidly pass the generation of animal models using RNAi. Especially exciting is the modification of animal genomes that can stretch to chromosomal deletions, inversions and translocations where multiple sgRNAs are utilized. Overall, the evolution of CRISPR-Cas9 continues to move towards a more efficient, dynamic and malleable tool at studying the power of gene manipulation in targeted therapies.
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