The Application of CRISPR/Cas9 Genome Editing to target the BLD2 locus in Chlamydomonas reinhardtii

Abstract

Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 genome editing is on the forefront as a tool to alter genomes. This new tool can target a specific gene of interest within the genome and produce insertions, deletions or frameshift mutations that result in partial or complete loss of function. However, in the unicellular green alga Chlamydomonas reinhardtii, application of CRISPR/Cas9 has had only limited success as a tool to study gene function (Jiang et al., 2014). The Johnson lab is working to develop vectors for the use of CRISPR/Cas9 in this model organism, and this thesis focuses on targeting the BLD2 locus in this cell for genomic editing. The BLD2 mutation locus is a divergent member of the tubulin family that encodes epsilon tubulin, an essential component of basal bodies/centrioles/microtubule organizing centers (MTOCs) (Dutcher et al., 2003). The BLD2 protein is conserved among algae, lower plants and mammalian cells; surprisingly, in Chlamydomonas bld2 loss-of-function mutants are viable but have severe structural defects in their MTOCs (Goodenough et al., 1975). In this thesis, CRISPR/Cas9 will be utilized to knock out a part of the epsilon tubulin encoding region to produce a phenocopy of the bld2 mutation. This research will provide information on the use of CRISPR/Cas9 in Chlamydomonas, while also targeting a gene whose product is of considerable interest