Fig. 5: Paired D10A Cas9n gene editing does not induce off-target editing and retains high on-target efficacy. | Nature Communications

Fig. 5: Paired D10A Cas9n gene editing does not induce off-target editing and retains high on-target efficacy.

From: Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease

Fig. 5: Paired D10A Cas9n gene editing does not induce off-target editing and retains high on-target efficacy.

a Schematic of CYBB exon 3 showing binding sites for sg3, sg4 and sg5 as well as distance between D10A Cas9n-induced nicks. b D10A Cas9n-mediated gene correction of the CYBB c.252 G > A variant in CYBB-HSPCs measured 4 days after nucleofection. c Proliferation and viability data of RNP + AAV-treated cells from (b). d RT-qPCR quantification of p21 (CDKN1A) mRNA levels measured 48 h after nucleofection. e Semi-solid methylcellulose-based CFU assay of gene-edited CYBB-HSPCs. f Off-target analysis of CYBB-HSPCs treated with sg3 HiFi Cas9 RNPs or sg3 + sg4 D10A Cas9n RNPs. Only off-targets that showed detectable editing in Fig. 4b were assayed. g Circos plots showing off-target-mediated translocations (OMTs) identified by CAST-seq. h Coverage plots from CAST-seq showing all reads mapped to a ±10 kb region of the on-target site. Deletions (DEL) are shown in orange, and inversions (INV) are shown in purple. Data represented as mean (n = 3 biological replicates) ± standard deviation. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. Source data are provided as a Source Data file.

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