Developing A Geminin - Fused Crispr/Cas9 System To Enhance The Genome Editing Efficiency

Abstract

The CRISPR/Cas9 system is a prominent tool that has recently become favored for precise genome modification. When doing gene editing, this system causes a DNA double-strand break (DSB). The DSB can be restored by the cell through either of the two pathways: non-homologous end-joining (NHEJ) route or homology-directed repair (HDR) route. The NHEJ pathway can randomly induce indels into the cleavage site, whereas through homologous repair, the HDR can introduce precise genomic modifications at the target sequence. However, the NHEJ pathway is dominant in the cell cycle since the HDR pathway is limited only to the late S and G2 phases. Therefore, finding mechanisms to enhance the HDR rate while simultaneously suppress the NHEJ pathway is integral in increasing the gene editing efficiency of the CRISPR/Cas9 system. One method of which is creating a cell cycle-dependent Cas9 by fusing it with Geminin, a DNA replication inhibitor, which appears at the S and G2 phases while not presents at other phases. In this thesis, we employed molecular cloning to insert a Geminin sequence into Cas9 plasmid to create a Cas9-Geminin fusion construct, which included the Geminin sequence in the correct orientation and frame on the Cas9 plasmid. The successful creation of the recombinant plasmid paves the way for future investigations into the functional impact of the Cas9-Geminin fusion in gene editing experiments.