CRISPR-Cas9 Plasmid Cloning Protocol

Cas9 and gRNA_Cloning vector used for the nucleofection experiments were purchased from Addgene (plasmids #41815 and #41824).³⁵ (link) The gRNA_Cloning vector was optimized for gRNA cloning and expression by inserting the partially missing U6 promoter and gRNA scaffold sequences into the SpeI and NdeI sites of the gRNA_Cloning vector.³⁵ (link) The EGFP-targeting sgRNAs obtained by annealing the BH001–BH004 and BH037–BH044 oligonucleotides (Table S1) were cloned into the modified gRNA_Cloning vector using the Golden Gate Protocol.³⁶ (link)
The dicistronic BFP/EGFP and BFP/CYBB lentiviral vectors were generated by first inserting EGFP- and CYBB cDNAs into MscI and SbfI sites of the TagBFP-expressing lentiviral vector -pHR’SINcPPT-SBW obtained from M. Grez.³⁷ (link) Subsequently, an ECMV-IRES was cloned into the MscI site between the BFP/EGFP or BFP/CYBB cDNAs.
EGFP mutations were introduced by inserting annealed synthetic oligonucleotides into the BstXI sites of EGFP (Oligos SFHR037, SFHR038, and SFHR046–SFHR055). CYBB mutations were introduced by standard site-specific mutagenesis using the BH137–BH144 primers (Table S1).
pLentiCRISPRv2 vectors containing the different sgRNAs were obtained by target-specific oligonucleotide annealing (Table S1) using the GoldenGate protocol.