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Updated: Jun 19, 2025

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

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Developing small Cas9 hybrids using molecular modeling.

Antoine Mangin^1,2, Vincent Dion^3,4, Georgina Menzies⁵

¹UK Dementia Research Institute at Cardiff University, Cardiff, CF24 4HQ, UK.

Scientific Reports

|July 26, 2024

Summary

This summary is machine-generated.

Researchers explored smaller Cas9 variants and hybrids to contract disease-causing CAG/CTG repeats for treating neuromuscular disorders. However, these engineered systems failed to achieve gene editing, indicating limitations in current in silico design for complex Cas9/sgRNA systems.

Keywords:

Binding energy Cas9 Gene editing Molecular dynamics

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Area of Science:

Gene editing technologies
Molecular biology
Biotechnology

Background:

CAG/CTG repeat contractions offer a therapeutic strategy for over 15 neuromuscular and neurodegenerative diseases.
Current methods using Streptococcus pyogenes Cas9 (SpCas9) face delivery challenges due to enzyme size, hindering clinical translation.

Purpose of the Study:

To investigate smaller Cas9 orthologues (SlugCas9, OgeuIscB) and novel Cas9/sgRNA hybrids for efficient CAG/CTG repeat contraction.
To overcome adeno-associated virus packaging limitations for in vivo gene editing applications.

Main Methods:

Screening of smaller Cas9 orthologues (SlugCas9, OgeuIscB) for repeat contraction activity.
Design and synthesis of Cas9/sgRNA hybrid systems using molecular dynamics and binding energy calculations.
In vitro testing of Cas9/sgRNA hybrids in human cells for gene editing efficiency.

Main Results:

SlugCas9 and OgeuIscB were ineffective in inducing CAG/CTG repeat contractions.
Developed Cas9/sgRNA hybrid pairs, despite in silico design, failed to edit target sequences in human cells.
In silico methods proved insufficient for designing functional, large-scale Cas9/sgRNA complexes.

Conclusions:

Smaller Cas9 orthologues and designed hybrids do not effectively contract CAG/CTG repeats.
Current computational approaches are limited in designing complex gene editing systems for therapeutic applications.
Further research is needed to develop viable delivery systems for treating repeat expansion diseases.