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Overview
Mutinfo:episomal CRISPR system for NR knockout |
Organism:Nannochloropsis oceanica (N. oceanica CCMP1779) |
Method:CRISPR/Cas |
Genes:jgi.p|Nanoce1779_2|590448 |
Plasmids:Addgene Article-28189686, Plasmid #99863, #101009, #100009, #99369, #100008, #99861, #99370, #98147 |
Promoter:Ribi |
Selection:Hygromycin |
Phenotypes:We present a one-vector episomal CRISPR/Cas9 system for N. oceanica that enables the generation of marker-free mutant lines. The CEN/ARS6 region from Saccharomyces cerevisiae was included in the vector to facilitate its maintenance as circular extrachromosal DNA. The vector utilizes a bidirectional promoter to produce both Cas9 and a ribozyme flanked sgRNA. This system efficiently generates targeted mutations, and allows the loss of episomal DNA after the removal of selection pressure, resulting in marker-free non-transgenic engineered lines. To test this system, we disrupted the nitrate reductase gene (NR) and subsequently removed the CRISPR episome to generate non-transgenic marker-free nitrate reductase knockout lines (NR-KO).
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