Pbs hsp70 cas9

For human cells, expression vector PX330 (Addgene plasmid 42230) encoding Cas9 and chimeric guide RNA was used (11 (link)). The LBR guides were cloned into expression vector pBluescript with the sgRNA cassette of PX330 and transfected into the K562 line stably transformed with Cas9. For Drosophila cells, Cas9 expression vector pBS-Hsp70-Cas9 (Addgene plasmid 46294) was used in combination with pU6-BbsI-chiRNA construct (Addgene plasmid 45946) (12 (link)). The sgRNAs were designed using CRISPR design (http://crispr.mit.edu/) (13 (link)) and CHOPCHOP (https://chopchop.rc.fas.harvard.edu/) (14 (link)).
The following sgRNA sequences were used:
For the cloning of individual DNA fragments from the edited GFP gene, PCR products were ligated in Zero Blunt vector (Invitrogen) using standard procedures.

Injections were conducted by Rainbow Transgenic Flies. The donor plasmid TAREhNU2G (507 ng/μL) was injected along with plasmid EGDhg2t (149 ng/μL), which provided gRNAs for transformation and was constructed previously [42 (link)], and pBS-Hsp70-Cas9 (442 ng/μL, Addgene plasmid #45945), which provided Cas9. A 10 mM Tris-HCl, 100 μM EDTA solution at pH 8.5 was used for the injection. Both lines containing split homing modification [26 (link)] and suppression [47 ] drives were generated in previous studies.

Gene knockout in OSCs using the CRIPSR/Cas9 system and isolation of knockout OSCs were performed essentially as described previously (Ishizu et al. 2015 (link)). In brief, OSCs were transfected with 0.2 μg of a plasmid expressing both pBS-Hsp70-Cas9 (Addgene, 46294) and the blasticidin resistance gene and 4 µg of two sgRNA-expressing plasmids (GM76 and E2) using Xfect transfection reagent (TaKaRa Clontech). After incubation for 24 h, blasticidin (Thermo Fisher Scientific, Inc., R210-01) was added to the culture medium at 50 µg/mL. The next day, 5.0 × 10³ cells were passaged in 3.5-cm dishes and allowed to grow in blasticidin-containing medium. During culturing, cells were washed with the medium to remove nonadherent dead cells. After 6–7 d of culture, 5.0 × 10³ cells were passaged in 6-cm dishes and allowed to grow in blasticidin-containing medium. After further incubation for a few days, colonies were picked and passaged in single wells of 24-well plates. The next day, these colonies were suspended with a Tip Strainer (Bel-Art) and allowed to grow to confluence. To detect the genomic deletion, genomic DNA was extracted using QuickExtract DNA extraction solution (Epicenter) following the manufacturer's protocol, and the genomic region flanking the CRISPR target site was amplified by PCR and then analyzed using MultiNa (Shimadzu).