Arriving at the perfect CRISPR system and how to troubleshoot for therapeutic development.
Over the past decade, CRISPR systems have revolutionized both genome editing and molecular diagnostics. This versatile technology, capable of targeting a variety of genetic sequences, has steadily progressed from enabling fundamental research through gene knockouts to powering largescale genetic screens, ultimately culminating in the 2023 FDA approval of Casgevy, the first CRISPR-Cas9 gene therapy for sickle cell disease. Beyond Cas9, other CRISPR enzymes like Cas12a and Cas13a have proven crucial in developing rapid diagnostic tools, as demonstrated during the COVID-19 pandemic. But to arrive at the perfect CRISPR system for a specific application, you may have to troubleshoot multiple parameters along the way. CRISPR editing requires a Casenzyme that cuts the nucleic acid (DNA or RNA) at a specific site that is designated by a guide RNA. Additional elementsinclude the PAM site, which has roles in Cas binding and initiating DNA unwinding. High quality Cas enzymes is an important aspect of a CRISPR experiment and for scaling up, it’s important that these enzymes produce consistent and reproducible results. Other parameters to consider are the gRNA and PAM sequence, how to best deliver the CRISPRsystem into your cells of interest, and the type of cut you are making (ex: Cas9 cuts dsDNA, Cas12a cuts dsDNA and RNA, and Cas13a cuts RNA).
In this white paper, we take a look at these challenges and how to troubleshoot them for therapeutic development.