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Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
Published on: February 26, 2019
Developing small Cas9 hybrids using molecular modeling
Antoine Mangin1,2, Vincent Dion3,4, Georgina Menzies5
1UK Dementia Research Institute at Cardiff University, Cardiff, CF24 4HQ, UK.
Abstract:
The contraction of CAG/CTG repeats is an attractive approach to correct the mutation that causes at least 15 neuromuscular and neurodegenerative diseases, including Huntington's disease and Myotonic Dystrophy type 1. Contractions can be achieved in vivo using the Cas9 D10A nickase from Streptococcus pyogenes (SpCas9) using a single guide RNA (sgRNA) against the repeat tract. One hurdle on the path to the clinic is that SpCas9 is too large to be packaged together with its sgRNA into a single adeno-associated virus. Here we aimed to circumvent this problem using the smaller Cas9 orthologue, SlugCas9, and the Cas9 ancestor OgeuIscB. We found them to be ineffective in inducing contractions, despite their advertised PAM sequences being compatible with CAG/CTG repeats. Thus, we further developed smaller Cas9 hybrids, made of the PAM interacting domain of S. pyogenes and the catalytic domains of the smaller Cas9 orthologues. We also designed the cognate sgRNA hybrids using molecular dynamic simulations and binding energy calculations. We found that the four Cas9/sgRNA hybrid pairs tested in human cells failed to edit their target sequences. We conclude that in silico approaches can identify functional changes caused by point mutations but are not sufficient for designing larger scale complexes of Cas9/sgRNA hybrids.
Insights
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.
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.
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