Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Generation of hypoimmunogenic induced pluripotent stem cells by CRISPR-Cas9 system and detailed evaluation for clinical application

doi: 10.1016/j.omtm.2022.05.010

Figure Lengend Snippet: Generation and evaluation of simultaneous knockout of the HLA-A, HLA-B, and CIITA genes in iPSCs by CRISPR-Cas9 (A) The design of a gRNA (HLA-A24-ex2g1) to simultaneously target the HLA-A and HLA-B genes, and another gRNA (CIITA-ex3g5) to target the CIITA gene. The genomic coordinate is based on hg19. (B) Histograms of the immunoreactivity intensity of the anti-HLA-A and anti-HLA-B antibodies for bulk genome-edited iPSCs. (C) The workflow of the production of genome-edited iPSC clones and their evaluation. (D) Sanger sequencing of the genome-edited iPSCs after single-cell cloning. Insertions or deletions at the target sites in the genome-edited iPSC clones were confirmed by electropherograms. (E) Size distribution of insertions or deletions at each target gene locus.

Article Snippet: Briefly, 0.3 × 10 6 iPSCs were transfected with an RNP (ribonucleoprotein) complex of 5 μg of GMP-grade recombinant Cas9 (Takara-bio, Shiga, Japan) and 1.25 μg of chemical synthesized gRNA (Ajinomoto Bio-Pharma) in 20 μL of Primary P3 buffer and electroporation condition program CA-137.

Techniques: Knock-Out, CRISPR, Clone Assay, Sequencing