Journal: Cell Reports Medicine
Article Title: Senescence and inflammation are unintended adverse consequences of CRISPR-Cas9/AAV6-mediated gene editing in hematopoietic stem cells
doi: 10.1016/j.xcrm.2025.102157
Figure Lengend Snippet: Anakinra treatment reduces the genotoxicity risk associated with the gene-editing procedure (A) Visualization of chromosomal translocation genome wide by CAST-Seq. (B and C) Copies of AAVS1 sequences per human genome 800 bp downstream to the target site in individual colonies generated by edited mPB HSPCs (B: n = 46, 93, 97, 93, and 85) or CB HSPCs (C: n = 48, 91, 92, 98, and 97) from 3 independent donors. LME, linear-mixed model. (D) Mutational rate of total variants in the indicated conditions from BM-derived CD34 + HSPCs of mice from one independent experiment from I ( n = 6, 6, 5, and 4); Kruskal-Wallis test. (E) Percentage of single-nucleotide variants (SNVs), deletions (DELs), and insertions (INSs) in BM-derived CD34 + HSPCs retrieved from mice in (D). n = 6, 6, 5, and 4; Kruskal-Wallis test; mean ± SEM. (F) Relative proportion (percentages) of SNV types in mice in (E). n = 6, 6, 5, and 4; Kruskal-Wallis test; mean ± SEM. (G) Percentage of variants with high (HIGH), moderate (MODERATE), or low (LOW) impact found in BM-derived CD34 + HSPCs retrieved from mice in (D) ( n = 6, 6, 5, and 4); Kruskal-Wallis test; mean ± SEM. (H and I) Quantification and Circos plots representing variants in clonal hematopoiesis (CH)-associated genes (H) or cancer-associated genes (I) ( n = 6, 6, 5, and 4); Kruskal-Wallis test. p values: ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001. If not indicated, results are not significant (ns). Unless otherwise specified, lines indicate median values. (−): HS/AAV6 condition.
Article Snippet: CB- or mPB-derived HSPCs were treated with 3.5 μg of p53 inhibitor ( GSE56 mRNA) co-electroporated with RNP complex, 50 ng/μL of IL-1 receptor antagonist Anakinra (Kineret, SOBI) and 25 μM InSolutionTM IKK-2 Inhibitor SC514 (Merck, 401485) administered immediately after the GE procedure.
Techniques: Translocation Assay, Genome Wide, Generated, Derivative Assay