Journal: The Plant journal : for cell and molecular biology
Article Title: Non-transgenic marker-free gene disruption by an episomal CRISPR system in the oleaginous microalga, Nannochloropsis oceanica CCMP1779
Figure Lengend Snippet: Development of an episomal CRISPR system. (A) The S. cerevisiae CEN/ARS6 region (ARS, red) was included in the pNOC-ARS-CRISPR construct for episomal maintenance. Guide sequences(orange) for nitrate reductase targets, with a 5’hammerhead ribozyme (HH),were fused to the gRNA scaffold to form the NR sgRNAs (sgNR). Ribozymes are highlighted in yellow. The sgNR1 and sgNR2were added to pNOC-ARS-CRISPR to form pNOC-ARS-CRISPR-sgNR1 and pNOC-ARS-CRISPR-sgNR2, respectively. N. oceanica is transformed with circular episomal CRISPR constructs. (B) Mutations in the two target sites in the NR genomic locus (Target 1 and Target 2) of N. oceanica ). Deleted nucleotides are represented with dashes and inserted nucleotides are shown in bold. Protospacer adjacent motifs (PAM sites) are underlined. (C) Immunoblot using an α-HA antibody of N. oceanica NR1-KO and NR2-KO lines producing Cas9-Nlux-HA from the CRISPR episome. Numbers on the left indicate size markers (KDa). (D) Growth curves after transfer from NH 4 to NO 3 containing medium of NR-KO frame-shifted lines (1D, 1H, 4G, B12) and NR2-IF B7 in-frame line, empty vector integrated CRISPR control lines (iEV), and wildtype (WT). (E) Episome rescue by E. coli transformation using equal quantities of DNA isolated from episomal (NR-KO) lines, integrated empty-vector (iEV), and wild-type (WT) N. oceanica lines. Values are the average colonies generated ± SE (NR-KOs n = 3 independent lines, WT n = 2 biological replicates, and iEV n = 2 independent lines). Equal quantities of DNA from NR-KO lines after treatment were used for E. coli transformation, and the resulting colonies counted (n = 3 independent lines). Exonuclease V (ExoV), ClaI endonuclease (ClaI), and ClaI endonuclease with Exonuclease V (ClaI+ExoV).
Article Snippet: For enzyme treatment of the DNA extracted from NR-KO lines, 2 μg of DNA was treated with 10 units of Exonuclease V (NEB) and/or 10 units of ClaI (NEB) in a 20 μl reaction for 1 hour at 37° C. Reactions were heat inactivated at 75° C for 30 min. An equal amount of DNA (500 ng) from enzyme treated samples, mock treated, and untreated samples were used to transform E. coli (DH5α high-efficiency efficiency, NEB).
Techniques: CRISPR, Construct, Transformation Assay, Plasmid Preparation, Isolation, Generated