Project description:Detecting genetic variants enables risk factor identification, disease screening and initiation of preventative therapeutics. However, current methods, relying on hybridization or sequencing, are unsuitable for point-of-care settings. In contrast, CRISPR-based-diagnostics offer high sensitivity and specificity for point-of-care applications. While these methods have predominantly been used for pathogen sensing, their utilization for genotyping is limited. Here, we report a multiplexed CRISPR-based genotyping assay using LwaCas13a, PsmCas13b, and LbaCas12a, enabling the simultaneous detection of six genotypes. We applied this assay to identify genetic variants in the APOL1 gene prevalent among African Americans, which are associated with an 8 to 30-fold increase in the risk of developing kidney disease. Machine learning facilitated robust analysis across a multi-center clinical cohort of more than 100 patients, accurately identifying their genotypes. Additionally, we optimized the readout using a multi-analyte lateral-flow assay demonstrating the ability for simplified genotype determination of clinical samples. Our CRISPR-based genotyping assay enables cost-effective point-of-care genetic variant detection due to its simplicity, versatility, and fast readout.
Project description:To assess gene expression by APOL1 genotypes in primary proximal tubule cells (PTCs), global gene expression (mRNA) levels were examined on Affymetrix HTA 2.0 arrays in primary PTCs cultured from non-diseased kidney in African Americans without CKD who underwent nephrectomy for localized renal cell carcinoma.
Project description:To elucidate pathways whereby apolipoprotein L1 gene (APOL1) G1 and G2 variants facilitate kidney disease in African Americans, human embryonic kidney cells (HEK293) were used to establish doxycycline-inducible (Tet-on) cell lines stably expressing reference APOL1 G0 and its G1 and G2 renal-risk variants. Illumina human HT-12-v4 arrays and Affymetrix HTA 2.0 arrays were employed to generate global gene expression data with doxycycline induction. Significantly altered pathways identified through bioinformatics involved mitochondrial function; results were validated using immunoblotting, immunofluorescence and functional assays. Global gene expression profiles were performed on HEK293 Tet-on G0, G1, G2 and empty vector cells with and without Dox induction using Illumina human HT-12 v4 arrays. Another independent gene expression array system, Affymetrix HTA 2.0, was used to verify the results of Illumina arrays. Pair-wise and pattern-based analyses were applied to detect the mostly impacted pathways due to overexpression and by APOL1 genotypes.
Project description:To elucidate pathways whereby apolipoprotein L1 gene (APOL1) G1 and G2 variants facilitate kidney disease in African Americans, human embryonic kidney cells (HEK293) were used to establish doxycycline-inducible (Tet-on) cell lines stably expressing reference APOL1 G0 and its G1 and G2 renal-risk variants. Illumina human HT-12-v4 arrays and Affymetrix HTA 2.0 arrays were employed to generate global gene expression data with doxycycline induction. Significantly altered pathways identified through bioinformatics involved mitochondrial function; results were validated using immunoblotting, immunofluorescence and functional assays.
Project description:To assess differential gene expression by APOL1 renal-risk (2 risk alleles) vs. non-risk (G0G0) genotypes in primary proximal tubule cells (PTCs), global gene expression (mRNA) levels were examined on Affymetrix HTA 2.0 arrays in primary PTCs cultured from non-diseased kidney in African Americans without CKD who underwent nephrectomy for localized renal cell carcinoma. To detect differentially expressed gene profiles attributable to APOL1 renal-risk genotypes, African American primary proximal tubule cells with two APOL1 renal-risk alleles (N=5) and lacking renal-risk alleles (N=25) were included in comparisons of global gene expression.
Project description:We describe the use of saturation genome editing to measure the effects of CARD11 variants on protein function, splicing and lymphoma cell survival. We find the results to predict the clinical effects of the variants.
Project description:Detecting genetic variants enables risk factor identification, disease screening, and initiation of preventative therapeutics. However, current methods, relying on hybridization or sequencing, are unsuitable for point-of-care settings. In contrast, CRISPR-based-diagnostics offer high sensitivity and specificity for point-of-care applications. While these methods have predominantly been used for pathogen sensing, their utilization for genotyping is limited. Here, we report a multiplexed CRISPR-based genotyping assay using LwaCas13a, PsmCas13b, and LbaCas12a, enabling the simultaneous detection of six genotypes. We applied this assay to identify genetic variants in the APOL1 gene prevalent among African Americans, which are associated with an 8-30-fold increase in the risk of developing kidney disease. Machine learning facilitated robust analysis across a multicenter clinical cohort of more than 100 patients, accurately identifying their genotypes. In addition, we optimized the readout using a multi-analyte lateral-flow assay demonstrating the ability for simplified genotype determination of clinical samples. Our CRISPR-based genotyping assay enables cost-effective point-of-care genetic variant detection due to its simplicity, versatility, and fast readout.
Project description:Gene sequence mutations may alter mRNA transcription, transcript stability, protein translation, protein stability and protein folding. Apolipoprotein L1 (APOL1) has two sets of sequence variants that are risk factors for kidney disease development, APOL1G1 (substitution mutation) and APOL1G2 (deletion mutation). Our present study focuses on the impact of these variants on APOL1 mRNA transcription and translation. APOL1 plasmids (EV, G0, G1 and G2) were transfected into human embryonic kidney (HEK) 293T cells. APOL1 variant expression was observed to be significantly lower than that of APOL1G0. Podocyte cell lines stably expressing APOL1 transgenes also showed lower levels of APOL1 expression of APOL1 variants (G1 and G2) compared with APOL1G0 by Western blotting and FACS analysis. The enhanced expression of GRP78 by podocytes expressing APOL1 variants would indicate endoplasmic reticulum (ER) stress. Bioinformatics evaluation using two different programs (MUPro and I-Mutant 2.0) predicted that APOL1 variants were less stable than APOL1G0. Concomitant with protein levels, APOL1 mRNA levels were also depressed following induction of APOL1 variant compared with APOL1G0 in both proliferating and differentiated podocytes. APOL1 mRNA transcript stability was tested after actinomycin D pulsing; APOL1G1 and APOL1G2 mRNAs transcript decayed 10-15% and 15-20% (within a period of 0.5-3 h) respectively. Our data suggest that down-regulated APOL1 protein expression in APOL1 variants is due to compromised transcription and decay of the APOL1 variant transcripts.