I have attempted various methods, including homozygous gene disruption and CRISPR-Cas9, but have not achieved the desired results. It appears that the gene of interest is situated in a nucleosome-rich region of the genome, complicating the process. I would greatly appreciate any assistance or insights from experts in the field.
Hello,
I do not perform knocking out, but here are some advices from publications and internet (Article Genome engineering in Saccharomyces cerevisiae using CRISPR-...
Article New CRISPR Mutagenesis Strategies Reveal Variation in Repair...
) ,CRISPR-Cas9 Optimization
- since your gene is in a nucleosome-rich region, consider using CRISPR-Cas9 strategies that involve nucleosome remodelling (CRISPR-DT)
- try using Cas9 D10A nickase mutant that creates single-strand breaks, which can reduce off-target effects and might improve efficiency in difficult regions
- TALENs might provide an alternative to CRISPR by allowing for more precise targeting and potentially better access to nucleosome-rich regions
- increase the length of homology arms in your donor DNA to improve HDR efficiency (arms of 1 kb or longer can enhance integration efficiency)
- use small molecules like RS-1, which enhance HDR efficiency by modulating DNA repair pathways
RNAi can be used to knock down gene expression if complete knockout proves difficult (promoter replacement strategies).
Histone Deacetylase Inhibitors like Trichostatin A (TSA) can help open up chromatin structure, making the target site more accessible for CRISPR or other gene editing tools.
I hope this information can help you.
Good luck ;-)
- Badges
- Science topic
- Similar topics
- Social Psychology
- Attitude