CRISPR Plasmid Construction for Genome Editing

pSPgRNA (#47108), pCMV-PE2 (#132775), pU6-Sp-pegRNA-GG-acceptor (#132777), and pU6-Sp-pegRNA_HEK3_CTT_ins (#132778) were obtained from Addgene (Watertown, MA). The U6-pegRNA vectors were constructed with pU6-Sp-pegRNA-GG-acceptor and the U6-nicksing sgRNA vectors were constructed with pSPgRNA. pegRNAs were constructed with pU6-Sp-pegRNA-GG-acceptor, as previously reported.^{3 (link)} Nicking sgRNAs were generated by T4 ligation of annealed oligos into the BbsI-digested pSPgRNA plasmid. The pegRNA and nicking sgRNA sequences for different targets in HEK293 cells and HEK293-rd12 cells are listed in Supplementary Tables S1 and S2. By in-fusion cloning of PCR amplicons to restriction enzyme-digested backbones, the N- and C-terminals of PE2 were produced. Every plasmid was confirmed by sequencing.

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She K., Liu Y., Zhao Q., Jin X., Yang Y., Su J., Li R., Song L., Xiao J., Yao S., Lu F., Wei Y, & Yang Y. (2023). Dual-AAV split prime editor corrects the mutation and phenotype in mice with inherited retinal degeneration. Signal Transduction and Targeted Therapy, 8, 57.

Publication 2023

pSPgRNA pCMV-PE2

Backbones Hek293 cells Ligation Oligos Plasmid Restriction enzyme Vectors

Corresponding Organization :

Other organizations : Sichuan University, West China Hospital of Sichuan University

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Variable analysis

independent variables

PSPgRNA (#47108)
PCMV-PE2 (#132775)
PU6-Sp-pegRNA-GG-acceptor (#132777)
PU6-Sp-pegRNA_HEK3_CTT_ins (#132778)

dependent variables

Efficiency of pegRNA and nicking sgRNA constructs in HEK293 cells and HEK293-rd12 cells

control variables

Sequencing of every plasmid to confirm their identity

positive controls

Not specified

negative controls

Not specified

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