A Genetic Engineering Toolbox for Marmosets (GETMarm): Development and optimization of genome editing and assisted reproduction techniques for marmoset models

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Project Summary The major goal of this grant proposal is to develop and refine germline genetic manipulation technologies for marmoset embryos to facilitate the generation of genetic engineered marmoset models for brain research. To increase the efficiency of generating genetic engineered marmoset models with desired DNA changes, one of the technologies we proposed to develop in the grant is preimplantation genetic testing (PGT). Briefly, after CRISPR editing in marmoset embryos, we culture the embryos in vitro and take either one embryonic cell (from 6-8 cell stage embryos) or 5-10 supporting cells (from 100-200 cell blastocysts). We will amplify the DNA region by PCR and sequence the DNA fragment to identify embryos with correct editing while the embryos are still in culture. We need to get the sequencing data withing 24 hours to ensure good embryo survival. Once we identify the embryos with correct editing, we will then implant the correct embryos to recipient marmosets. Currently, the fastest commercial sequencing service is 48 hours and even that is not guaranteed. We have also checked with Broad sequencing platform service and their turnaround time is about two weeks and they only do large batch sequencing. Fortunately, Thermo-Fisher has recently released a bench-top sequencer that is easy to use and we could get the sequencing done within hours. This equipment will greatly facilitate our PGT and dramatically increase our successful rate of genome editing in marmosets.