Figure 2.
Efficacy and specificity of genome editing at the RAG2 locus using universal correction strategy. (A) Schematics of gene-targeted integration of codon- coRAG2 and an expression cassette. The coRAG2 sequence is under the control of an endogenous promoter. (B) Overview of genome-editing outcomes. (C) Percent coRAG2-GTed in healthy donor (HD)purified HSPCs from fresh CB and frozen PB. Each circle represents a unique human HSPC donor. Genome-targeted (GTed) integration of coRAG2 was quantified by digital-droplet polymerase chain reaction (ddPCR). (D) Frequency of cells with 1 (monoallelic) or 2 (biallelic) alleles targeted as a function of the virus’ MOI. Analysis was performed on single cells sorted on methylcellulose plates (n = 5 at 5000 MOI; n = 4 at 2500 MOI; and n = 2 at 1250 MOI). Bars ± SEM (E) OT analysis using RAG2-SCID (c.296C>A and c.1342C>A) patient-derived HSPCs. Next-generation sequencing of 48 COSMID predicted OT sites in edited-only (RNP-sgRNA guide no. 3 and HiFi Cas9 nuclease) or electroporated-only (mock, nucleofected without RNP) cells. Shown INDELs reads for on-target (RAG2 gene, white circle) and OT sites below the limit of detection (gray circles) and above the limit of detection (green circles). PacBio-based analysis overview of (F) quantification of perfect and imperfect homologous recombination events using PacBio sequencing. Marked as “wild-type∗” are events that retained the wild-type protospacer adjacent motif sequence even after perfect HR repair. (G) A total of 85.5% of the bulk non-GT alleles have INDELs and 14.5% are wild-type (WT) alleles. The brown bar represents the background signal from sequencing error. (H) An overview of the INDEL spectrum in non-GT alleles identified short deletions (∼13 bp) as the predominant events. Each dot represents the frequency of a unique PacBio long-read size bin. Red box shows ≥100 bp deletions in (I) top histogram. Blue box shows ≥100 bp insertions in (I) bottom histogram. Percent total frequency of ≥100 bp deletions is 4.2 and of ≥100 bp insertions is 4.0 (supplemental Figure 4D). SNP, single nucleotide polymorphism.

Efficacy and specificity of genome editing at the RAG2 locus using universal correction strategy. (A) Schematics of gene-targeted integration of codon- coRAG2 and an expression cassette. The coRAG2 sequence is under the control of an endogenous promoter. (B) Overview of genome-editing outcomes. (C) Percent coRAG2-GTed in healthy donor (HD)purified HSPCs from fresh CB and frozen PB. Each circle represents a unique human HSPC donor. Genome-targeted (GTed) integration of coRAG2 was quantified by digital-droplet polymerase chain reaction (ddPCR). (D) Frequency of cells with 1 (monoallelic) or 2 (biallelic) alleles targeted as a function of the virus’ MOI. Analysis was performed on single cells sorted on methylcellulose plates (n = 5 at 5000 MOI; n = 4 at 2500 MOI; and n = 2 at 1250 MOI). Bars ± SEM (E) OT analysis using RAG2-SCID (c.296C>A and c.1342C>A) patient-derived HSPCs. Next-generation sequencing of 48 COSMID predicted OT sites in edited-only (RNP-sgRNA guide no. 3 and HiFi Cas9 nuclease) or electroporated-only (mock, nucleofected without RNP) cells. Shown INDELs reads for on-target (RAG2 gene, white circle) and OT sites below the limit of detection (gray circles) and above the limit of detection (green circles). PacBio-based analysis overview of (F) quantification of perfect and imperfect homologous recombination events using PacBio sequencing. Marked as “wild-type∗” are events that retained the wild-type protospacer adjacent motif sequence even after perfect HR repair. (G) A total of 85.5% of the bulk non-GT alleles have INDELs and 14.5% are wild-type (WT) alleles. The brown bar represents the background signal from sequencing error. (H) An overview of the INDEL spectrum in non-GT alleles identified short deletions (∼13 bp) as the predominant events. Each dot represents the frequency of a unique PacBio long-read size bin. Red box shows ≥100 bp deletions in (I) top histogram. Blue box shows ≥100 bp insertions in (I) bottom histogram. Percent total frequency of ≥100 bp deletions is 4.2 and of ≥100 bp insertions is 4.0 (supplemental Figure 4D). SNP, single nucleotide polymorphism.

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