# Developing small Cas9 hybrids using molecular modeling

## TL;DR

Researchers tried to create smaller Cas9 hybrids to treat diseases caused by CAG/CTG repeats but found that in silico methods alone are insufficient for designing functional CRISPR systems.

## Contribution

The study shows that in silico methods can identify functional changes but are not enough for designing complex Cas9/sgRNA hybrids.

## Key findings

- Smaller Cas9 orthologues like SlugCas9 and OgeuIscB were ineffective in inducing CAG/CTG repeat contractions.
- Cas9/sgRNA hybrid pairs designed using molecular modeling failed to edit target sequences in human cells.
- In silico approaches are insufficient for designing larger scale CRISPR complexes.

## 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.