Naslov
Active fusions of Cas9 orthologs

Autori
Josipović, Goran ; Zoldoš, Vlatka ; Vojta, Aleksandar

Izvornik
Journal of biotechnology (0168-1656) 301 (2019); 18-23

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
Cas9 ortholog ; Cas9 fusion ; sgRNA ; DNMT3A ; Epigenome editing

Sažetak
Many recent epigenetic studies utilize the advantages of CRISPR/dCas9 based tools in linking certain epigenetic modification with gene expression regulation. Various multifactorial diseases often contain changed epigenetic signatures at many loci, so tools for simultaneously targeting different loci would significantly facilitate the understanding of disease pathogenesis. We tested different dCas9 orthologs (dCjCas9, dNmCas9, dSt1Cas9, dFnCas9, dSaCas9 and dSpCas9) in C-terminal fusion with DNMT3A effector domain to find candidates that potentiate effector domain to perform its function at the target site. We demonstrated that nuclear localization signals (NLS) at both termini of fusion constructs is crucial for both proper nuclear import of such large constructs as well as for maximization of targeted DNA methylation activity. We identified SpCas9, SaCas9 and CjCas9 as potential candidates for the fusion constructs. With further optimization of the SaCas9 ortholog, due to less complex PAM requirements in contrast to CjCas9, we showed that N-terminal fusion with DNMT3A (dSaCas9- DNMT3A) is optimal to exert targeted DNA methylation activity comparable to the dSpCas9- DNMT3A construct. N-terminal fusions showed better results for both Cas9 orthologs, SaCas9 and SpCas9, so it can be used as universal approach for linking different effector domains in order to obtain highly active fusions.

Izvorni jezik
Engleski

Znanstvena područja
Biologija, Biotehnologija u biomedicini (prirodno područje, biomedicina i zdravstvo, biotehničko područje)

POVEZANOST RADA