Programmable tracking of RNA molecules in live cells via CRISPR/Cas9

[Read the Manuscript Here] Opinion by Arit Ghosh, Ph.D.

Current advancements in molecular biology have revolutionized molecular medicine. With the advent of a novel gene editing technology named CRISPR/Cas9 gene therapy seems like a reality after all. CRISPR/Cas9 has become an integral tool in molecular biology and has been widely adopted by several research labs around the world. CRISPR-Cas9 has become the staple of almost every research group conducting genome engineering to investigate function of wide variety of genes. Due to the advantages the methodology holds over gene therapy or RNAi has led to CRISPRi mediated gene editing becoming the herald for novel therapeutics. Important differences when comparing with conventional gene editing via Zinc Finger proteins and TALEN technology or even RNAi mediated gene silencing are mainly due superior target design simplicity, higher efficiency, ability to introduce multiplexed mutation. For a detailed technical analysis of CRISPR Cas9 technology – this is an rigorous annual review on the topic – (CRISPR/Cas9 in Genome Editing and Beyond).

In this study, the authors demonstrate that CRISPR/Cas9 genome editing can be used to target RNA molecules and allow tracking of endogenous RNA transcripts in living cells in a manner free from genetically encoded tags thereby involving less modifications of the target transcripts. The nuclease-inactive Streptococcus pyogenes CRISPR/Cas9 can bind RNA in a nucleic-acid programmed manner which allows endogenous RNA tracking in living cells. The authors utilize the guide RNA and PAMmer sequence in the RNA molecules to direct this specificity. The work suggests that RNA fate and turnover can potentially be tracked in a more efficient manner compared to current methods such as just conventional RNA-FISH (Flourescence In Situ Hybridization). The overlap analysis of recombinant Cas9 (RCas9) and FISH makes this method a superior one compared to conventional methods. As mentioned in this work – “simple RNA targeting afforded by RCas9 could support the development of sensors that recognize specific healthy or disease-related gene expression patterns and reprogram cell behavior via alteration of gene expression or concatenation of enzymes on a target RNA”. Indeed delivery of Cas9 in vivo is a subject of current research (Cationic lipid-mediated delivery) and combining such methods with current oligonucleotide chemistries (RNA Targeting Therapeutics) could support the efforts. In the cell – RNA goes through various processing steps such as alternative splicing, nuclear export, subcellular localization and even sequestration during stress into P-bodies and stress granules. A proof of principle study extending this work could deal with RNA/stress granule formation. A lot of these processes such as stress granule formation are indications of early onset of diseases in the cellular environment and tracking such granule formation and connecting the dots to the genes involved in RNA sequestration could help in early detection.

#CRISPR #RNA #CellBiology

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