Knocking out bzip14 gene using CRISPR/Cas9 and analyzing the mutant line in response to induced ER stress in Marchantia polymorpha

Accumulation of unfolded and mis-folded proteins cause ER stress. In response to ER stress, the unfolded protein response (UPR) is triggered by ER transmembrane sensor. UPR, a set of integrated signaling pathways, designed to restore ER homeostasis.  Two bZIP transcriptional factors regulate the UPR pathway in Marchantia Polymorpha: bZIP14 and bZIP7. In this study, bzip14 mutant lines were generated using CRISPR/Cas9. In this order, the bZIP14 DNA sequence was identified by Blastp in Marchantia.info, a database website for M.  polymorpha. Exons are implied to find out the proper sgRNAs as candidates in http://crispor.tefor.net/. Among all suggested sgRNAs, three of them were selected as candidates. sgRNAs were introduced into pMpGE013, a specific CRISPR/Cas9 vector in M. polymorpha. After transformation to agrobacteria GWB303+pSOUP, it was transformed into the plants. DNA was extracted from plants regenerated in selective media containing hygromycin, amplified using PCR by specific primers, and sent to sequencing. Eight out of 11 lines that were sequenced properly showed deletion and insertion in the DNA sequence. One mutant line was chosen for phenotypic assay and a survival test in the media containing tunicamycin, ER stress inducer. Our result showed that there are two isoforms of bZIP17 and bZIP28 in Arabidopsis thaliana, as homologs with bZIP14 in M. polymorpha. Also, a significant difference (p-value ≤ 0.05) was observed between bzip14 mutant and wild type under ER stress conditions.