Synthetic mismatches enable specific CRISPR-Cas12a-based detection of genome-wide SNVs tracked by ARTEMIS

Figures

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  Figure 1. Outcomes of ARTEMIS for Cas12a-based detection of SNVs (A) A total of 21-nt Cas12a target sites are preceded by a 5′ tetranucleotide PAM sequence. The first five PAM-proximal nucleotides of the target site are complementary to the seed sequence of the crRNA. (B) ARTEMIS identifies how many PAM sequences are found genome-wide and what percentage of these are not coinciding with common SNVs (minor allele frequency > 1%) or have overlapping seed sequences. (C) ARTEMIS finds that of all found pathogenic SNVs, roughly 12% colocalize within the Cas12a seed sequence. (D) Amount of found cancer-associated SNVs within Cas12a seed regions per gene. An overview of targetable SNVs per chromosome can be found in Figure S1B. (E) A common oncogenic SNV found is BRAF c.T1799A, which is a missense mutation at PAM+1, resulting in protein variant p.V600E.

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  Figure 2. Introducing synthetic mismatches in the crRNA yields single-nucleotide fidelity BRAF p.V600E detection (A) crRNAs for p.V600E and wild-type (WT) BRAF detection (KR105 and KR122, respectively) were used in CRISPRdx reactions containing either V600E or WT activator. The variant of interest causing p.V600E is located at PAM+1. (B–D) Engineering the crRNA by introducing an SM at (B) PAM+2, (C) PAM+3, or (D) PAM+4 significantly improves the on-target activity. (E) Fluorescence ratios at different time points demonstrate improved single-nucleotide specificity for BRAF p.V600E detection with limited or no loss of activity. Ratios were calculated by dividing fluorescence signal from reactions spiked with V600E by values measured in reactions spiked with V600 WT. Error bars display the SD of the mean value of triplicate experiments. Activators were added to a final reaction concentration of 50 pM.

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  Figure 3. Cas12a distinguishes between heterozygosity and homozygosity for BRAF p.V600E alleles (A) Schematic overview of the derivation of cfDNA from conditioned mammalian cell culture medium. After isolation of shed DNA fragments, nucleic acid amplification enables Cas12-based detection and collateral cleavage of a quenched (Q) fluorescence (F) probe. (B–D) SKmel28 (B), (C) WM9, and (D) VU1131 serve as characterized control cell lines for which the BRAF status and zygosity are known.24 Slopes were calculated from the displayed solid regression lines. (E) Regression slopes from CRISPRdx reactions on a melanoma cell line panel with unknown BRAF statuses (Mel25, Mel28, Mel78, Mel79, Mel84, and Mel88). (F) Comparison between fluorescence endpoint ratio at 3 h and regression slope ratio on a logarithmic scale. Regressions were done over 3 h. Ratios were calculated by division normalization for KR125 fluorescence or slope. Reactions display triplicate experiments, and error bars indicate SEMs.

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  Figure 4. PAM-distal synthetic mismatches do not confer increased specificity for variant recognition within the seed region (A) Overview of 8 different crRNAs with single or dual synthetic mismatches against Mel78 (BRAFL597P homozygous) for the detection of BRAFV600E or BRAFV600 at PAM+1. Positions in the target sequence relative to the PAM site have been indicated every five bases. (B) Fluorescence endpoint values after 3 h of incubation. (C) Fluorescence ratio scores calculated from endpoint fluorescence values at 3 h of incubation, plotted on a logarithmic scale. Ratio scores were calculated by normalizing reactions with BRAFV600E-detecting crRNAs to reactions with BRAFV600-detecting crRNAs. Error bars display the SD of the mean value of triplicate experiments.

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  Figure 5. Cas12a identifies BRAFV600E-positive sequences in human plasma samples (A and B) Raw fluorescence data of CRISPRdx using (A) KR125 and (B) KR121 over 3 h. Culture-derived cfDNA from WM9, a characterized heterozygous BRAFV600E cell line, was used as control input material. Error bars display SD of the mean of triplicate experiments. (C) Endpoint fluorescence values after 3 h of incubation. All three replicate values are shown, together with error bars displaying the SD. Where relevant, results from a two-way ANOVA comparison between tested samples and the water control have been indicated as ns (non-significant) or as ∗∗∗∗p < 0.0001. (D) Overview of the BRAFV600E status of the tested samples as determined by CRISPRdx and NGS, compared with the measured S-100B levels.

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  Figure 6. crRNA-based optimization of CRISPRdx on pathogenic seed region mutations in BRCA2, TP53, and ALDH2 (A) Graphic overview of the BRCA2E1953∗ target site in exon 11 and a panel of six crRNA spacers. (B) The 3-h endpoint fluorescence ratio per crRNA, calculated from separate CRISPRdx reactions with spiked synthetic mutant or WT BRCA2 dsDNA. (C) Graphic overview of the TP53V272M target site in exon 8 and a panel of eight crRNA spacers. (D) The 3-h endpoint fluorescence ratio per crRNA, calculated from separate CRISPRdx reactions with spiked synthetic mutant or WT TP53 dsDNA. (E) Graphic overview of the ALDH2E504K target site in exon 12 and a panel of four crRNA spacers. (F) The 3-h endpoint fluorescence ratio per crRNA, calculated from separate CRISPRdx reactions with spiked synthetic mutant or WT ALDH2 dsDNA. All target site graphics annotate the GC content of the seed region and of the entire spacer. The 5′ crRNA hairpins are not displayed in the graphic, and synthetic mismatches are indicated with red in italics. Fluorescence ratios were calculated through dividing reactions with mutant DNA (mut) by reactions with WT DNA (wt). Fold changes between fluorescence ratios have been…