Cas12a-based one-pot SNP detection with high accuracy

Abstract

CRISPR-Cas12a based one-pot detection system has been used in nucleic acid detection and diagnosis. However, it is not sensitive enough to distinguish single nucleotide polymorphisms (SNP), which has greatly restricted its application. To overcome these limitations, we engineered a LbCas12a variant with enhanced sensitivity against SNP, named seCas12a (sensitive Cas12a). SeCas12a-based one-pot SNP detection system is a versatile platform that could use both canonical and non-canonical PAM, and was almost not limited by mutation types to distinguish SNPs located between position 1 to 17. The use of truncated crRNA further improved SNP specificity of seCas12a. Mechanistically, we found only when the cis-cleavage was at low level between 0.01min^-1 and 0.0006 min^-1, a good signal-to-noise ratio can be achieved in one-pot test. SeCas12a-based one-pot SNP detection system was applied to detect pharmacogenomic SNPs in human clinical samples. Of thirteen donors tested in two different SNPs, the seCas12a mediated one-pot system could faithfully detect the SNPs in 30 min with 100% accuracy.

Graphical abstract

Fig. 1

Schematic diagram of seCas12a-based SNP detection system. (A) Schematic illustration of seCas12a-based SNP detection method. In one-pot SNP test, RPA and Cas12a-based detection were performed simultaneously. Allele specific crRNA was added to each reaction and fluorescence readout was used to deduct the SNP (B) Schematic of crRNA design. CrRNA G/681 G or crRNA A/681A refers to crRNA fully complementary to CYP2C19∗2681 G or A allele respectively. Signal or noise refers to fluorescence produced by matched or unmatched genotype respectively.

Fig. 2

Genetically engineered LbCas12a to allow SNP detection in one-pot test. (A) Heatmap of end point fluorescence signals (collected at 60 min). First round of screening of LbCas12a variants was tested in one-pot SNP detection of CYP2C19∗2. CrRNA G was used and tested in CYP2C19 ∗2 SNP bearing G or A allele. (B) Representative real-time fluorescence curves of LbCas12a variants with enhanced sensitivity. Fluorescence signals were read with a 1-min ​intervals (λ_ex = 485 nm; λ_em = 528 nm). (C) Heatmap of end point fluorescence signals using crRNA A in CYP2C19∗2 SNP. (D) Real-time fluorescence curve of LbCas12a variants in (C). Fluorescence signals were read with a 1-min ​intervals (λ_ex = 485 nm; λ_em = 528 nm).

Fig. 3

Optimization of crRNA can improve sensitivity in one-pot SNP detection. (A-B) Real-time fluorescence curve (A) and endpoint comparison (B) of WT or seCas12a based one-pot SNP detection targeting CYP2C9∗3 (42,614 A/C) SNP. Experiments were performed independently for three times and data were plotted as mean ​± ​S.D. (C) In vitro cis-cleavage activity of WT or seCas12 with indicated crRNA targeting CYP2C9∗3 (42,614 A/C) SNP. (D) Cis-cleavage kinetic analysis of Cas12a-based SNP detection. Corresponding signal-to-noise ratio was calculated using endpoint fluorescence signals measured at 30 min in one-pot reaction. (E) Correlation analysis of cleavage kinetics and one-pot SNP signal-to-noise ratios.

Fig. 4

SeCas12 mediated one-pot SNP detection is flexible with PAM selection. (A) Canonical (gray) and non-canonical (blue) PAM of LbCas12a in human genome. (B) Real-time fluorescence curve of non-canonical (5’-TCCA-3’) and canonical PAMs (5’-TTTA-3’) in one-pot reactions. Truncated crRNA A (19 nt) or crRNA C (19 nt) targeting CYP2C9∗3 42,614 A or C allele respectively were used (n ​= ​3, mean ​± ​S.D.). (C-D) Real-time fluorescence curve (C) and endpoint (D) representation of the effects of SNP positions on one-pot sensitivity. 19 nt crRNA A targeting 42,614 A was used.N = 3, mean ± S.D. (E) Real-time fluorescence curve and endpoint representation of the effects of SNP types on one-pot sensitivity. 19 nt crRNA A and crRNA C targeting 42,614 A or C allele were used. N = 3, mean ± S.D. (F-G) Real-time fluorescence curve (F) and endpoint (G) representation of substrates with indicated SNP ratios. N = 3, mean ± S.D.

Fig. 5

Application of seCas12-mediated one-pot test in clinical samples. (A) Detection of CYP2C9∗3 (42,614 A/C) SNP using truncated crRNAs in 13 donors tested. Truncated crRNA A (19 nt) and crRNA C (19 nt) could faithfully detect the respective genotype.(B) Detection of CYP2C19∗2 (681 G/A) allele using full length crRNAs in 13 donors. CrRNA G (20 nt) and crRNA A (20 nt) could faithfully detect the respective genotype. NC is a negative control without any DNA substrate. Data are mean ± S.D. (n = 3).