Figure 2.
Figure 2. LMO2 intron 1 indels are predominantly monoallelically activating, and CRISPR/Cas9-mediated knockout of the PF-382 mutant allele downregulates LMO2 expression. (A) LMO2 expression as determined by qRT-PCR in human sorted thymic subsets, primary patient samples with LMO2 intron 1 indels, and the wild-type Jurkat cell line. P < .002 for samples A1, A2, A3, A9, and P6 vs double negative and double positive by two-tailed t test. Primary patient samples were assessed for the absence of biallelic TCR-γ deletion (ABD), of which patient sample A4 (orange bar) exhibited ABD, whereas all other patients were non-ABD. (B) The informative single nucleotide polymorphism (SNP) (rs3740617) was amplified in 4 patient samples and the DU.528 cell line from both genomic DNA (gDNA) and complementary DNA (cDNA) templates to infer monoallelic expression. To do this, if one chromatogram peak is detected at a heterozygous SNP within the cDNA, the expression can be interpreted as coming from one allele. (C) Quantification of the number of reads mapped to the wild-type (WT) or mutant (MUT) allele where 54 of 56 reads and 85 of 85 reads mapped to the mutant alleles for DU.528 and PF-382, respectively. (D) Firefly luciferase activity after renilla and no-insert vector normalization for patient-derived indels. Data shown are from ≥3 independent experiments performed in triplicate. Values are mean ± standard deviation and P values were calculated by a two-tailed Student t test. (E) The yellow highlighted sequence is the target region for the CRISPR/Cas9 guide RNA. Aligned sequences are from CRISPR/Cas9-edited PF-382 single-cell clones showing the associated genomic edits generated. Red sequences are inserted sequences, blue are altered, and dashes represent deleted bases. Underlined region shows the presence of the native and mutant MYB binding sites. (F) Gene expression of LMO2 for each PF-382 clone, as determined by qRT-PCR. Data are expressed as fold change relative to the mean expression of the unedited clones in arbitrary units (AU). Clones are labeled as “unedited” when CRISPR/Cas9 did not edit the region targeted by the guide RNA and “edited” when successful targeting led to the formation of an indel. *P ≤ .05, **P ≤ .01, ***P ≤ .001, and ****P ≤ .0001. NOI, no-insert control; T/C, T to C single nucleotide polymorphism.

LMO2 intron 1 indels are predominantly monoallelically activating, and CRISPR/Cas9-mediated knockout of the PF-382 mutant allele downregulates LMO2 expression. (A) LMO2 expression as determined by qRT-PCR in human sorted thymic subsets, primary patient samples with LMO2 intron 1 indels, and the wild-type Jurkat cell line. P < .002 for samples A1, A2, A3, A9, and P6 vs double negative and double positive by two-tailed t test. Primary patient samples were assessed for the absence of biallelic TCR-γ deletion (ABD), of which patient sample A4 (orange bar) exhibited ABD, whereas all other patients were non-ABD. (B) The informative single nucleotide polymorphism (SNP) (rs3740617) was amplified in 4 patient samples and the DU.528 cell line from both genomic DNA (gDNA) and complementary DNA (cDNA) templates to infer monoallelic expression. To do this, if one chromatogram peak is detected at a heterozygous SNP within the cDNA, the expression can be interpreted as coming from one allele. (C) Quantification of the number of reads mapped to the wild-type (WT) or mutant (MUT) allele where 54 of 56 reads and 85 of 85 reads mapped to the mutant alleles for DU.528 and PF-382, respectively. (D) Firefly luciferase activity after renilla and no-insert vector normalization for patient-derived indels. Data shown are from ≥3 independent experiments performed in triplicate. Values are mean ± standard deviation and P values were calculated by a two-tailed Student t test. (E) The yellow highlighted sequence is the target region for the CRISPR/Cas9 guide RNA. Aligned sequences are from CRISPR/Cas9-edited PF-382 single-cell clones showing the associated genomic edits generated. Red sequences are inserted sequences, blue are altered, and dashes represent deleted bases. Underlined region shows the presence of the native and mutant MYB binding sites. (F) Gene expression of LMO2 for each PF-382 clone, as determined by qRT-PCR. Data are expressed as fold change relative to the mean expression of the unedited clones in arbitrary units (AU). Clones are labeled as “unedited” when CRISPR/Cas9 did not edit the region targeted by the guide RNA and “edited” when successful targeting led to the formation of an indel. *P ≤ .05, **P ≤ .01, ***P ≤ .001, and ****P ≤ .0001. NOI, no-insert control; T/C, T to C single nucleotide polymorphism.

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