Isolate 2 plasmid mini kit

Two randomly isolates of the expected size were extracted from agarose gels by GeneJET Gel Extraction Kit (Thermo Scientific, USA) and inserted into the pGEM T-Easy vector T-A cloning kit (Promega, USA). 5 µl of 2X ligation buffer was put into an Eppendorf tube contained l µl of pGEM T-Easy vector, 2 µl of insert DNA, l µl of T4 ligase enzyme, and 1 µl of µl RNase free water in a total volume of 10 µl. The ligation protocol was carried out overnight at +4 °C. The resulting recombinant plasmids were transferred into the E. coli JM109 competent strain (Promega) by electric shock.
Transformed bacteria were planted in solid LB medium and transformed white colonies including insert DNA were selected after overnight and planted in liquid LB medium including ampicillin. Finally, recombinant plasmids, bearing the cloned viral CP gene, were purified using the ISOLATE II Plasmid Mini Kit (Bioline, Germany), sent to the Sentebiolab company (Ankara/Turkey) for sequencing and sequences were recorded in the GenBank.

Single guide RNA (sgRNA) sequences for non-targeting control and ACE2 were taken from the Weissman Human Genome-wide CRISPRa-v2 library (Addgene #83978). LRRC15 sgRNA sequences and additional ACE2 sgRNA sequences were taken from the Human CRISPR activation pooled library set A (Addgene #92379). Sense and antisense strands for each sequence were ordered as DNA oligonucleotides (IDT) with 5' overhangs of 5'-CACC-3' on the sense strand oligonucleotide and 5'-AAAC-3' on the antisense strand oligonucleotide. Oligonucleotides were annealed at 4°C for 16 h and pXPR-502 (Addgene #96923) was digested with Esp3I (ThermoFisher Scientific, ER0451) or BsmBI-v2 (New England Biolabs). sgRNA DNA oligonucleotide duplexes were ligated into the digested pXPR-502 backbone using T4 ligase (New England Biolabs) and incubated at 4°C overnight. NEB 10-beta competent E. coli (New England Biolabs) were transformed with 100 ng of each sgRNA construct by heat-shock, plated onto LBagar plates (Life Technologies) containing ampicillin (Sigma-Aldrich) and grown at 37°C. Individual colonies were picked, expanded in Luria broth (Life Technologies) supplemented with ampicillin and amplified constructs were harvested using either ISOLATE II Plasmid Mini Kit (Bioline) or PureYield Plasmid Maxiprep System (Promega Corporation).

A strong PCR positive band selected was gel-purified by DNA isolation kit following the supplier's specifications (ISOLATE II PCR and Gel Kit, BIOLINE) and employed as material in the later stages of the studies. This fragment was ligated into a prokaryotic cloning vector (pGEM T-Easy vector, Promega) using T4 ligation enzyme followed by transferred into competent cells (E. coli JM109 strain) using micropulser. A transformed clone that confirmed positive by colony PCR tests was selected and subsequently sequenced by NGS (Sentebiolab/Ankara) after plasmid purification from bacterial cells (ISOLATE II Plasmid Mini Kit, BIOLINE). Plasmids containing 16S rRNA inserts were stored at -80°C for further use. 16S rRNA Iğdır sequence associated with phytoplasma was trimmed from recombinant vector sequences with UGENE program and used for further analysis during the study. Pathogenic location and sequence identity of Iğdır sequence were determined by using the BLASTN search program on the NCBI site, and the similarity coefficient was calculated by web-supported iPhyclassifier software, a useful tool for the assignment of phytoplasma strains.

The pOCacta-CFP plasmid was purified with ISOLATE II Plasmid Mini Kit (Bioline, England). The purified pOCacta-CFP plasmid was adjusted to 50 μg.mL -1 in 1× TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and microinjected into one-cell stage O. curvinotus embryos by following Vu et al., 2014 (link). The average amount of DNA introduced to each embryo was approximately 50 pg, as suggested by Cho et al. (2011) (link). Microinjected embryos were kept in the incubator at 28 °C. The dead and unfertilised embryos were counted and removed.
CFP expression embryos [from 1-to 14-day post fertilisation (dpf)] were observed using Nikon Eclipse TS100 fluorescence microscope with B-2A filter (excitation filter wavelengths: 450-490 nm; emission filter wavelength: 520 nm). The image of CFP-positive embryos was photographed using Nikon Eclipse TS100 digital camera. The O. curvinotus embryos that expressed CFP in the skeletal muscle were selected and maintained for adult growth.