Investigation of the properties and activity of DfCas9 and DsCas9 nucleases in eucaryotic cells

Abstract

This study is focused on the two novel nucleases of the CRISPR/Cas9 family, which were found in bacterial genomes of DfCas9 (Defluviimonas sp) и DsCas9 (Demequina sediminicola). Discovery of these nucleases was part of the results of a joint study conducted by BIOCAD together with Skoltech Institute of Science and Technology and Saint-Petersburg Polytechnical University (SPPU) under a grant agreement with the Department of Science and Education of Russian Federation (Agreement number 14.606.21.0006 from September, 26th 2017). Under the agreement the nucleases DfCas9 and DsCas9 were characterized in vitro by Skoltech and SPPU.

Based on the aforementioned results, in this study we characterized the genome-modifying nuclease activity of these enzymes in a mammalian cell line HEK293. Specifically, we created genetic constructs designed to express the nucleases DsCas9 and DfCas9 together with the necessary guide RNA molecules (sequences of the guide RNAs were described previously) [1]. We demonstrated expression of the nucleases on a protein level, as well as activity of DfCas9 at the VEGF2 locus in HEK293 cells. The theoretical study was conducted by analyzing international and national literature. The experimental part was performed with a restriction-ligation cloning method, transient transfections, Western blot protein detection method, and a T7 nuclease-based method of detection of heteroduplex double-stranded DNA.

Keywords

gene therapy / gene editing / CRISPR/Cas9 / DsCas9 / DfCas9 and SpCas9 / genomic nucleases / nuclease activity and specificity / T7 endonuclease / genome modifications

References

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RIGHTS & PERMISSIONS

Vlasova Y.V., Madera D.A., Gershovich P.M.