Gabrielle Cournoyer; Hannah Porter

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Publication Date



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 protometal growth. In order to study the roles of specific CSLDs in P. patens development, potential quadruple KO mutants for PpCSLD3/4/5/8? were generated by CRISPR/Cas9 mutagenesis. The goal of this project was to screen potential ppcsld3/4/5/8? KO lines for PpCSLD8 gene deletions using competition-based PCR (cbPCR) and perform sequence analysis on lines positive for gene deletions. A total of 21 potential ppcsld3/4/5/8? KO lines were screened and 7/21 were positive for potential PpCSLD8 deletions. The seven lines with potential deletions were sequenced and it was determined that all lines had the same six base pair insertion located just upstream of the potential deletion site. We hypothesize that this insertion was created due to off-target editing from the CRISPR-Cas9 PpCSLD4 vector, when the PpCSLD4 gene was knocked out. This insertion most likely prevented the PpCSLD8 gene from being knocked out using our current strategy.

Faculty Mentor

Christos Dimos, Ph.D.

Academic Discipline

College of Arts & Sciences



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