To construct CRISPR/Cas plasmids for HBB targeting, sgRNA oligos were synthesized by IDT and cloned into pX330 (Addgene: plasmid #42230) and pX335 (Addgene: plasmid #42335), respectively, following the Zhang lab protocol (https://www.addgene.org/crispr/zhang/ ).
To construct CRISPR/Cas double nicking plasmids for HBB targeting, human 7SK promoter-driven truncated sgRNA sequences49 (link) were synthesized by IDT and cloned into the KpnI site of one sgRNA-bearing pX335 vector by Gibson Assembly (NEB).
The CRISPR/Cas components of pX330-HBB-T2 were transferred to the shuttle vector pM7.7 via PciI and NotI sites to derive pM7.7-330- HBB-T2.
To construct CRISPR/Cas double nicking plasmids for HBB targeting, human 7SK promoter-driven truncated sgRNA sequences49 (link) were synthesized by IDT and cloned into the KpnI site of one sgRNA-bearing pX335 vector by Gibson Assembly (NEB).
The CRISPR/Cas components of pX330-HBB-T2 were transferred to the shuttle vector pM7.7 via PciI and NotI sites to derive pM7.7-330- HBB-T2.
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