Site-specific gene knock-in and bacterial phytase gene expression in Chlamydomonas reinhardtii via Cas9 RNP-mediated HDR.

ABSTRACT: In the present study, we applied the HDR (homology-directed DNA repair) CRISPR-Cas9-mediated knock-in system to accurately insert an optimized foreign bacterial phytase gene at a specific site of the nitrate reductase (NR) gene (exon 2) to achieve homologous recombination with the stability of the transgene and reduce insertion site effects or gene silencing. To this end, we successfully knocked-in the targeted NR gene of Chlamydomonas reinhardtii using the bacterial phytase gene cassette through direct delivery of the CRISPR/Cas9 system as the ribonucleoprotein (RNP) complex consisting of Cas9 protein and the specific single guide RNAs (sgRNAs). The NR insertion site editing was confirmed by PCR and sequencing of the transgene positive clones. Moreover, 24 clones with

SUBMITTER: Zadabbas Shahabadi H 

PROVIDER: S-EPMC10235511 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature