Related Experiment Video

Updated: Jun 7, 2025

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

Published on: April 26, 2017

Deciphering Cas9 specificity: Role of domain dynamics and RNA:DNA hybrid interactions revealed through machine

Gayatri Panda¹, Arjun Ray¹

¹Department of Computational Biology, Indraprastha Institute of Information Technology, New Delhi, India.

International Journal of Biological Macromolecules

|November 20, 2024

Abstract:

CRISPR/Cas9 technology is widely used for gene editing, but off-targeting still remains a major concern in therapeutic applications. Although Cas9 variants with better mismatch discrimination have been developed, they have significantly lower rates of on-target DNA cleavage. This study compares the dynamics of the highly specific Cas9 from Francisella novicida (FnCas9) to the commonly used SpCas9. Using long-scale atomistic Gaussian accelerated molecular dynamic simulations and machine learning techniques, we deciphered the structural factors behind FnCas9's higher specificity in native and off-target forms. Our analysis revealed that Cas9's cleavage specificity relies more on its domain rearrangement than on RNA:DNA heteroduplex shape, with significant conformational variations in Cas9 domains among off-target forms, while the RNA:DNA hybrid showed minimal changes, especially in FnCas9 compared to SpCas9. REC1-REC3 domains contacts with the RNA:DNA hybrid in FnCas9 acted as critical discriminator of off-target effects playing a pivotal role in influencing specificity. In FnCas9, allosteric signal transmission involves the REC3 and HNH domain, bypassing REC2, leading to a superior efficiency in information transmission. This study offers a quantitative framework for understanding the structural basis of elevated specificity, paving the way for the rational design of Cas9 variants with improved precision and specificity in genome editing applications.

Insights

Francisella novicida Cas9 (FnCas9) exhibits higher specificity than SpCas9 due to domain rearrangements, not RNA:DNA shape. This discovery aids in designing more precise gene editing tools.

Area of Science:

Molecular Biology
Biochemistry
Genomics

Background:

CRISPR/Cas9 gene editing is crucial but limited by off-targeting concerns.
Existing Cas9 variants with improved specificity often show reduced on-target cleavage efficiency.

Purpose of the Study:

To compare the dynamics of Francisella novicida Cas9 (FnCas9) with standard SpCas9.
To elucidate the structural determinants of FnCas9's enhanced specificity using advanced simulations and machine learning.

Main Methods:

Long-scale atomistic Gaussian accelerated molecular dynamics simulations.
Machine learning techniques to analyze structural dynamics and specificity.
Comparative analysis of FnCas9 and SpCas9 in native and off-target states.

Main Results:

FnCas9 specificity is primarily governed by domain rearrangement, not RNA:DNA heteroduplex shape.
Off-target binding induces significant conformational changes in Cas9 domains, with minimal impact on the RNA:DNA hybrid, especially for FnCas9.
REC1-REC3 domain interactions with the RNA:DNA hybrid are key discriminators of off-target effects in FnCas9.
Allosteric signal transmission in FnCas9 involves REC3 and HNH domains, bypassing REC2, enhancing information transfer efficiency.

Conclusions:

FnCas9 possesses superior specificity due to distinct domain dynamics and allosteric signaling pathways.
Understanding these structural factors provides a quantitative framework for developing highly specific Cas9 variants.
This research facilitates the rational design of next-generation genome editing tools with improved precision.

Read full analysis

Keywords:

Accelerated molecular dynamics Allostery CRISPR/Cas9 Feature-importance Machine-learning Markov-state modeling Off-target activity UMAP dimension reduction etc

More Related Videos

CRISPR-Mediated Reorganization of Chromatin Loop Structure

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Published on: September 14, 2018

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Published on: May 25, 2018

Related Concept Videos

Conservation of Protein Domains Over Different Proteins

Conservation of Protein Domains Over Different Proteins

Protein domains are small structurally independent units that are part of a single amino acid chain. Although these domains are often structurally independent, they may rely on synergistic effects to perform their functions as part of a larger protein. Protein domains may be conserved within the same organism, as well as across different organisms.
A limited set of protein domains often duplicate and recombine during evolution. These domains can be organized in different combinations to...

Conserved Binding Sites

Conserved Binding Sites

Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally...

Cooperative Binding of Transcription Regulators

Cooperative Binding of Transcription Regulators

Transcriptional regulators bind to specific cis-regulatory sequences in the DNA to regulate gene transcription. These cis-regulatory sequences are very short, usually less than ten nucleotide pairs in length. The short length means that there is a high probability of the exact same sequence randomly occurring throughout the genome. Since regulators can also bind to groups of similar sequences, this further increases the chances of random binding. Transcriptional regulators form...

CRISPR

CRISPR

Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...

Bacterial RNA Polymerase

Bacterial RNA Polymerase

Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...

siRNA - Small Interfering RNAs

siRNA - Small Interfering RNAs

Small interfering RNAs, or siRNAs, are short regulatory RNA molecules that can silence genes post-transcriptionally, as well as the transcriptional level in some cases. siRNAs are important for protecting cells against viral infections and silencing transposable genetic elements.
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the...

Articles linked to this work by shared authors, journal, and citation graph.

KinaseDB: an integrated multi-omics platform for the human kinome with disease-specific target prioritization.

Nucleic acids research·2026

LiMeNEx: an interactive webserver on lipid biochemical gene-regulatory network.

Nucleic acids research·2026

Phospholipids that Plug the Pores of Cholesteryl Ester Transfer Protein Control Its Triglyceride Transfer.

Computational and structural biotechnology journal·2026

Molecular Insights into Anabaenopeptin-Mediated Inhibition of Protein Tyrosine Phosphatase B in the Mycobacterium tuberculosis Complex.

ACS omega·2026

QKI ensures splicing fidelity during cardiogenesis by engaging the U6 tri-snRNP to activate splicing at weak 5' splice sites.

bioRxiv : the preprint server for biology·2025

Ser/Thr phosphorylation of Mycobacterium tuberculosis type II RelK toxin by PknK destabilizes TA interaction and interferes with toxin neutralization.

mBio·2025

See all related articles