Journal: Kidney360

Article Title: High-Fidelity CRISPR-Cas12a–Based Apolipoprotein L1 Genotyping

doi: 10.34067/KID.0000000871

Figure Lengend Snippet: Canonical PAM sequences are not required for CRISPR-Cas12a–mediated cis -cleavage of APOL1 DNA target. (A) Schematic diagram of interaction between Cas12a (represented in gray) and the APOL1 G1 risk allele wildtype sequence. The APOL1 target DNA strand is depicted in blue and the nontarget DNA strand in green. The crRNA is depicted in orange. The endonuclease cis- cleavage of the DNA target generating staggered ends is indicated by red triangles. Signal amplification is represented by the multiple-turnover Cas12a-mediated trans -cleavage of the FAM-labeled reporter probe, F-TTATT-Q. (B) Gel images of CRISPR-Cas12a–mediated cis -cleavage reaction products after electrophoretic separation on Agilent D1000 ScreenTapes. Reactions conducted in the absence of crRNA guides (lanes 1–4) show the intact 246-bp gBlock gene fragments (arrow labeled “Target”) corresponding to G0-A (wildtype—A) and G1-G (risk allele—G) lacking an endogenous PAM sequence (G0-A noPAM and G1-G noPAM, lanes 1 and 3, respectively) or with base substitutions to generate PAM sequences six bases proximal to the A or G single-nucleotide variant of the G1 allele (G0-A PAM and G1-G PAM, lanes 2 and 4, respectively). These four gBlock gene fragments were incubated with a G0 -A (lanes 5–8) or G1 -G (lanes 9–10) crRNA guide before electrophoresis. The bracketed arrows labeled “Products” indicate cis -cleavage products of approximately 180 and 60 bp. The sizes of the electronic DNA ladder (bp) produced by the TapeStation Analysis software is indicated on the left. (C) Fluorescence detection of CRISPR-Cas12–mediated trans -cleavage of FAM-labeled reporter probe. Fluorescence intensities of unquenched FAM-labeled reporter probe, following Cas12a-mediated trans -cleavage, were measured for each reaction every minute over a 1-hour incubation at 37°C. Reaction mixtures were prepared with a G0- A crRNA guide (green symbols) or without a crRNA guide (red symbols) and synthetic DNA gene fragments for each of the single-nucleotide variants, either lacking a PAM sequence (circles, G0-A no PAM; triangles, G1-G no PAM) or with artificial PAM sequences (squares, G0-A PAM; inverted triangles, G1-G PAM). APOL1 , apolipoprotein L1; CRISPR, clustered regularly interspaced short palindromic repeats; crRNA, CRISPR RNA; FAM, 6-carboxyfluorescein; PAM, protospacer adjacent motif.

Article Snippet: Residual DNA samples from deidentified human subjects representing all possible APOL1 genotypes obtained for proficiency testing of Arkana's Clinical Laboratory Improvement Amendments (CLIA)-approved APOL1 genotyping assay were used.

Techniques: CRISPR, Sequencing, Amplification, Labeling, Variant Assay, Incubation, Electrophoresis, Produced, Software, Fluorescence

Journal: Kidney360

Article Title: High-Fidelity CRISPR-Cas12a–Based Apolipoprotein L1 Genotyping

doi: 10.34067/KID.0000000871

Figure Lengend Snippet: Evaluation of crRNA guides for discrimination between APOL1 G1 risk alleles in trans -DNA cleavage assays. A series of 12 staggered, 17-nt crRNA guides targeting the APOL1-G0 (wildtype) allele on both the coding and noncoding strands were evaluated for their ability to discriminate between G0-A (wildtype) and G1-G (risk allele) synthetic gBlock APOL1 gene sequences. Depicted are the fluorescence intensity measurements of four crRNAs that revealed robust Cas12-mediated trans -DNase activity when incubated in the presence of the G0-A (green symbols) synthetic DNA target or but not with the G1-G (red symbols) target. The ratio of the fluorescence intensities measured after 30 minutes (vertical bar) indicated the highest discrimination with the G0-A/F5 (squares), followed by the G0-A/F6 (triangles), G0-A/R2 (inverted triangles), and G0-A/F3 (circles). DNase, deoxyribonuclease.

Article Snippet: Residual DNA samples from deidentified human subjects representing all possible APOL1 genotypes obtained for proficiency testing of Arkana's Clinical Laboratory Improvement Amendments (CLIA)-approved APOL1 genotyping assay were used.

Techniques: Fluorescence, Activity Assay, Incubation

Journal: Kidney360

Article Title: High-Fidelity CRISPR-Cas12a–Based Apolipoprotein L1 Genotyping

doi: 10.34067/KID.0000000871

Figure Lengend Snippet: Both fluorescence and NALF assays could be used for APOL1 genotyping DNA isolated from patient peripheral blood samples. (A) Representative fluorescence-based CRISPR-Cas12a–mediated APOL1 genotyping assays of gDNA isolated from two patient samples. Genomic DNA was amplified with primers targeting a portion of the APOL1 gene encompassing the two kidney risk alleles. The amplified DNA was then incubated with Cas12a and a FAM-labeled reporter probe in the presence of one of four crRNA guides targeting the wildtype alleles (green symbols) or risk variants (red symbols). The fluorescence intensities measured from the reactions with crRNAs targeting the single-nucleotide variants (A or G) are depicted with circles, and the crRNAs targeting the 6-bp indel (I or D) are depicted with squares. For each patient sample, the percentage of fluorescence signal obtained after 30 minutes of incubation (vertical bar) at each allele with the indicated crRNA is listed in the table below the graph along with the resulting genotype call. (B) Representative CRISPR-Cas12a–mediated NALFs of gDNA isolated from two patient samples. Genomic DNA was amplified with primers targeting a portion of the APOL1 gene encompassing the two kidney risk alleles. The amplified DNA was then incubated with Cas12a and a FAM- and biotin-labeled reporter probe in the presence of one of four crRNA guides targeting the wildtype alleles or risk variants. The reactions were then diluted with running buffer and applied to NALF test cassettes. After 15 minutes, the development of the control and test lines were visually evaluated and photographed. The development of the control line (C) indicates the test strips functioned properly. The presence of a lower test line (T) indicates the lack of trans -cleavage of the reporter probe, while the absence of a lower test lines indicates the trans -ssDNAse activity of Cas12a had been activated by the engagement of the targeting crRNA in the reaction. (C) Model of NALF results. Cas12a is incubated with a DNA sequence to be interrogated, a target-specific crRNA guide, and a FAM- and biotin-labeled nonspecific ssDNA reporter sequence. If the crRNA guide engages with the target DNA sequence, Cas12a ssDNAse activity is activated resulting in cleavage of the reporter probe. Cas12a reaction mixtures are applied to NALF test cassettes and are transported along the membrane by capillary action. Intact reporter probes (negative reactions) are captured at the test line using an anti-FAM antibody and visualized through an interaction between streptavidin-coated gold nanospheres and the biotin moiety of the reporter probe. By contrast, after Cas12a-meditated cleavage of the reporter probe (positive reactions), only the 5′-portion of the reporter probe is retained at the test line and no signal is detected because the streptavidin-coated gold nanospheres are not captured. A signal detected at the control line represents a second gold conjugate that is not affected by the presence of either the FAM or biotin moieties of the reporter probe, but signifies that the NALF test cassette components were successfully transported along the membrane during the incubation period. gDNA, genomic DNA; NALF, nucleic acid lateral flow; ssDNA, single-stranded DNA; ssDNAse, ssDNA nuclease.

Article Snippet: Residual DNA samples from deidentified human subjects representing all possible APOL1 genotypes obtained for proficiency testing of Arkana's Clinical Laboratory Improvement Amendments (CLIA)-approved APOL1 genotyping assay were used.

Techniques: Fluorescence, Isolation, CRISPR, Amplification, Incubation, Labeling, Control, Activity Assay, Sequencing, Membrane