Farbod Azaripour Masooleh—McMaster Life Sciences 2025
Gene Editing has been a frequently discussed topic with its increasing importance, as new technologies continue to improve it. CRISPR is one such gene editing technique that has revolutionized the field by making it more precise and easier. This has made scientists hopeful about the possibility of correcting disease-causing genes to prevent genetic disorders. The VI CRISPR-Cas effector, Cas13b, targets designated RNAs directly. The combination of Cas13b and ADAR2 adenosine deaminase domain with rational protein engineering has resulted in a more efficient enzyme. This has made efficient and specific RNA depletion of mammalian cells possible. This system is known as RNA Editing for Programmable A to I Replacement (REPAIR). This system can edit full-length transcripts carrying pathogenic mutations, not limited to specific sequences. To minimize the system and facilitate viral delivery, REPAIR is being modified to increase its specificity. This gives scientists a reliable RNA-editing platform with broad applicability for research studies, therapeutics, and biotechnology advancements. To test this system in humans, REPAIRv1 was developed to correct disease-causing G→A mutations in nucleotides.
Figure 1: measuring the flexibility of the sequences for editing RNA using REPAIRv1. SOURCE: Science
Substantial editing was achieved at 33 sites with an efficiency rate of 28%. The REPAIR system enables multiplex editing of multiple disease-causing variants. The dCas13b platform, designed for programmable RNA binding, can also be used for live transcript imaging, splicing modification, targeted localization of transcripts, RNA-binding protein pulldown, and epitranscriptomic modifications.
Figure 2: This image shows the use and effect of REPAIRv1 on repairing a G→A mutation. SOURCE: Science
The base conversions that scientists are able to achieve by using REPAIR are restricted to using adenosine in order to create inosine. But, combining dCas13 with other RNA editing domains, can help with editing cytidine to uridine. In addition, for relaxing the substrate preference so that cytidine can be targeted, mutagenesis of ADAR can be used. This grants more specificity from the duplexed RNA substrate requirement so that it can aid C to U editors.
In conclusion, as technology advances, the equipment used for scientific purposes gets more precise and accurate. CRISPR, as one such tool, holds great promise for curing genetic diseases and improving millions of lives in the near future.
Cox DB, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, et al. RNA editing …..with CRISPR-CAS13. Science. 2017Nov24;358(6366):1019–27.