Kerrina Whelan, Kayleigh Kearney

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The moss Physcomitrella patens is an attractive model organism due to its small genome and dominant haploid phase. P. patens has eight genes that form a family known as the Cellulose Synthase-Like Ds (CSLDs). A previous study using global knockdown of the entire PpCSLD family by RNA interference (RNAi) showed a decrease in protonemal tip growth, indicating that PpCSLDs play a role in protonemal growth. In order to study the roles of specific CSLDs in P. patens development, potential double KO mutants for PpCSLD5/8 were generated by CRISPR/Cas9 mutagenesis. The goal of this project was to screen potential ppcsld5/8 KO lines for gene deletions using PCR. A total of 59 potential ppcsld5/8 KO lines were screened and 4/59 were positive for PpCSLD5 deletions, while 1/59 was positive for a PpCSLD8 deletion. None of the potential ppcsld5/8 KO lines screened were positive for both PpCSLD5 and PpCSLD8 gene deletions. The high number of potential ppcsld5/8 KO lines reporting as wild type could be a result of the CRISPR/Cas9 system only cutting at one target site. This would result in minor indel events that cannot be detected by conventional PCR. As a result, Competition-based PCR (cbPCR) assays are being designed to look for indels in these potential ppcsld5/8 KO lines.

Faculty Mentor

Christos Dimos, PhD

Academic Discipline

BS - Biology



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