CRISPR-Cas9 Genome Editing in Cells

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.