[Paper Review] Gene and RNA Editing: Methods, Enabling Technologies, Applications, and Future Directions

Mohammed Aledhari, Mohamed Rahouti|arXiv (Cornell University)|Sep 1, 2024

RNA regulation and diseaseBiochemistry, Genetics and Molecular Biology3 citations

TL;DR

This paper reviews gene and RNA editing technologies, emphasizing CRISPR-based methods like Cas13 for RNA editing and Cas9 for DNA editing, highlighting their roles in treating genetic diseases. It concludes that RNA editing offers a safer, reversible alternative to permanent gene editing, with future potential in cellular biocomputing and diagnostics.

ABSTRACT

Gene and RNA editing methods, technologies, and applications are emerging as innovative forms of therapy and medicine, offering more efficient implementation compared to traditional pharmaceutical treatments. Current trends emphasize the urgent need for advanced methods and technologies to detect public health threats, including diseases and viral agents. Gene and RNA editing techniques enhance the ability to identify, modify, and ameliorate the effects of genetic diseases, disorders, and disabilities. Viral detection and identification methods present numerous opportunities for enabling technologies, such as CRISPR, applicable to both RNA and gene editing through the use of specific Cas proteins. This article explores the distinctions and benefits of RNA and gene editing processes, emphasizing their contributions to the future of medical treatment. CRISPR technology, particularly its adaptation via the Cas13 protein for RNA editing, is a significant advancement in gene editing. The article will delve into RNA and gene editing methodologies, focusing on techniques that alter and modify genetic coding. A-to-I and C-to-U editing are currently the most predominant methods of RNA modification. CRISPR stands out as the most cost-effective and customizable technology for both RNA and gene editing. Unlike permanent changes induced by cutting an individual's DNA genetic code, RNA editing offers temporary modifications by altering nucleoside bases in RNA strands, which can then attach to DNA strands as temporary modifiers.

Motivation & Objective

• To compare and contrast the mechanisms, benefits, and limitations of gene editing versus RNA editing in treating genetic disorders.
• To evaluate the role of CRISPR systems, particularly Cas13 for RNA editing and Cas9 for gene editing, in enabling precise, customizable genetic interventions.
• To assess current challenges in RNA editing detection, including low sequencing depth, false positives, and multimapping issues in tRNA and lncRNA analysis.
• To explore future applications of RNA editing as a safer therapeutic approach and its potential integration with cellular computing via engineered biocomputers.
• To identify key technological and computational bottlenecks in high-throughput sequencing and bioinformatics pipelines for RNA editing detection.

Proposed method

• Utilizes CRISPR-Cas systems, specifically Cas9 for DNA editing and Cas13 for RNA editing, to target and modify specific genetic sequences.
• Reviews A-to-I and C-to-U editing as the most prevalent RNA modification methods, focusing on their enzymatic mechanisms and biological impact.
• Analyzes high-throughput sequencing (HTS) and direct RNA sequencing for detecting RNA editing events, noting limitations in read depth and sensitivity.
• Examines challenges in read alignment and fragment boundary detection due to multimapping in tRNA and low-expression lncRNAs and miRNAs.
• Discusses computational pipelines for processing large-scale RNA-seq data, emphasizing the need for automated, accurate, and scalable annotation tools.
• Explores emerging biocomputing applications, such as CRISPR-based dual-core processors in human cells, using modified Cas9 as a digital half-adder.

Experimental results

Research questions

• RQ1 How do RNA editing and gene editing differ in mechanism, safety, and therapeutic applicability, particularly in treating genetic diseases?
• RQ2 What are the key technical limitations of current RNA sequencing technologies in detecting RNA editing events accurately and sensitively?
• RQ3 Why is RNA editing considered a safer alternative to permanent gene editing, and how can it be leveraged for future therapies?
• RQ4 How can CRISPR-based biocomputers be engineered to perform computational functions within human cells for diagnostics and treatment?
• RQ5 What are the major challenges in detecting low-abundance non-coding RNAs (e.g., lncRNAs, miRNAs) and how can they be overcome?

Key findings

• RNA editing, particularly via A-to-I and C-to-U modifications, offers a reversible and safer alternative to permanent gene editing, reducing off-target risks.
• CRISPR-Cas13 is a highly customizable and cost-effective tool for RNA editing, enabling transient modifications without altering the genome.
• Current RNA sequencing yields only ~1 million reads compared to over 6 million for DNA sequencing, limiting sensitivity and accuracy in RNA editing detection.
• False positives remain a significant challenge in HTS-based RNA editing detection, especially in protein-coding and non-coding RNAs with low expression.
• Multimapping of tRNA and lncRNA sequences to multiple genomic locations introduces ambiguity in determining the true origin of editing events.
• Future biocomputing applications, such as CRISPR-based dual-core processors in human cells, demonstrate the potential to integrate computing logic into cellular systems for advanced diagnostics.