Biology subjects

Chilamkurthy, R.

Publications and source records attributed to Chilamkurthy, R..

4 recordsLinked to original sources

bioRxiv2026

Chemical control of 2'-hydroxyl-dependent Cas9 target engagement enables CRISPR RNA ribose replacement

Advanced CRISPR-based therapies benefit from CRISPR RNA (crRNA) with high nuclease resistance and enhanced drug-like properties, which is primarily achieved through chemical replacement of the RNA ribose moiety. However, for gene editing enzymes like CRISPR-Cas9 a handful of residues cannot be replaced with chemical ribose analogues, limiting the scope of therapeutic strategies. The mechanism underlying this restriction has remained unclear. Here, using nucleic acid chemistry, biochemistry, cryo-EM, and molecular dynamics simulations, we show that the ribose 2-hydroxyl group at specific crRNA residues is required to achieve a conformational state competent for Cas9 target DNA binding. Based on the mechanistic principles uncovered, we combined site-specific phosphorothioate linkage chemistry with ribose replacement chemistry to restore binding and activity, resulting in high Cas9 editing efficiency and fidelity with a ribose-free crRNA. This study offers novel mechanistic insight and crRNAs with full chemical stabilization, making rational design of guide RNAs with complete nuclease protection for CRISPR-based medicines possible.

biochemistry↗

bioRxiv2025

A guide RNA repeat checkpoint steers CRISPR-Cas9 catalysis

A widely adopted CRISPR-Cas9 modification is fusion of the naturally occurring two-component dual guide RNA (dgRNA) to create an artificial single guide RNA (sgRNA). However, mechanistic and functional differences between dgRNA and sgRNA have not been systematically explored. By investigating the activity of these two guide architectures, we discover a guide RNA repeat checkpoint (GRC) that senses the structure and dynamics of the guide repeat region. The GRC coordinates with other checkpoint mechanisms known to recognize spacer-target R-loop fidelity, together licensing target cleavage. Based on these principles, dgRNA and sgRNA properties could be combined into guide repeat-truncated sgRNAs (grtRNAs) and paired with high-fidelity Cas9 variants to further reduce off target editing. A mechanism that helps govern Cas9 catalysis via guide RNA structure sensing and communicates with other checkpoints offers a previously unappreciated path for understanding and further improving gene editing outcomes.

biochemistry↗

bioRxiv2021

High Throughput Nanopore Sequencing of SARS-CoV-2 Viral Genomes from Patient Samples

In late 2019, a novel coronavirus began spreading in Wuhan, China, causing a potentially lethal respiratory viral infection. By early 2020, the novel coronavirus, called SARS-CoV-2, had spread globally, causing the COVID-19 pandemic. The infection and mutation rates of SARS-CoV-2 make it amenable to tracking movement and evolution by viral genome sequencing. Efforts to develop effective public health policies, therapeutics, or vaccines to treat or prevent COVID-19 are also expected to benefit from tracking mutations of the SARS-CoV-2 virus. Here we describe a set of comprehensive working protocols, from viral RNA extraction to analysis using online visualization tools, for high throughput sequencing of SARS-CoV-2 viral genomes using a MinION instrument. This set of protocols should serve as a reliable how-to reference for generating quality SARS-CoV-2 genome sequences with ARTIC primer sets and next-generation nanopore sequencing technology. In addition, many of the preparation, quality control, and analysis steps will be generally applicable to other sequencing platforms.

genomics↗

bioRxiv2021

Emergence and Evolution of a Prevalent New SARS-CoV-2 Variant in the United States

Genomic surveillance can lead to early identification of novel viral variants and inform pandemic response. Using this approach, we identified a new variant of the SARS-CoV-2 virus that emerged in the United States (U.S.). The earliest sequenced genomes of this variant, referred to as 20C-US, can be traced to Texas in late May of 2020. This variant circulated in the U.S. uncharacterized for months and rose to recent prevalence during the third pandemic wave. It initially acquired five novel, relatively unique non-synonymous mutations. 20C-US is continuing to acquire multiple new mutations, including three independently occurring spike protein mutations. Monitoring the ongoing evolution of 20C-US, as well as other novel emerging variants, will be essential for understanding SARS-CoV-2 host adaptation and predicting pandemic outcomes.

genomics↗