Elucidating the Role of CRISPR/Cas in Single-Step Isothermal Nucleic Acid Amplification Testing Assays

Abstract

Developing assays that combine CRISPR/Cas and isothermal nucleic acid amplification has become a burgeoning research area due to the novelty and simplicity of CRISPR/Cas and the potential for point-of-care uses. Most current research explores various two-step assays by appending different CRISPR/Cas effectors to the end of different isothermal nucleic acid amplification methods. However, efforts in integrating both components into more ideal single-step assays are scarce, and poor-performing single-step assays have been reported. Moreover, lack of investigations into CRISPR/Cas in single-step assays results in incomplete understanding. To fill this knowledge gap, we conducted a systematic investigation by developing and comparing assays that share the identical recombinase polymerase amplification (RPA) but differ in CRISPR/Cas12a. We found that the addition of CRISPR/Cas12a indeed unlocks signal amplification but, at the same time, impedes RPA and that CRISPR/Cas12a concentration is a key parameter for attenuating RPA impediment and ensuring assay performance. Accordingly, we found that our protospacer adjacent motif (PAM)-free CRISPR/Cas12a-assisted RPA assay, which only moderately impeded RPA at its optimal CRISPR/Cas12a concentration, outperformed its counterparts in assay design, signal, sensitivity, and speed. We also discovered that a new commercial Cas12a effector could also drive our PAM-free CRISPR/Cas12a-assisted RPA assay and reduce its cost, though simultaneously lowering its signal. Our study and the new insights can be broadly applied to steer and facilitate further advances in CRISPR/Cas-based assays.

Figure 1.

Designing and evaluating three comparable assays to elucidate the role of CRISPR/Cas12a in single-step assays. (A) The three assays share a common RPA reaction with the same pair of RPA primers, RPA reagents (e.g., recombinase, single-stranded DNA binding protein, polymerase), and DNA target. (B) The CRISPR/Cas12a-free RPA assay detects RPA amplicons through a fluorogenic exo probe—internally labeled with a fluorophore (F) and a quencher (Q) around a tetrahydrofuran (THF) residue and terminally labeled with a 3′ blocker—that hybridizes to its complementary strand of the RPA amplicon, gets cleaved at the THF residue by exonuclease III (the additional enzyme in the RPA reaction mix of this assay), and yields a fluorescence signal. (C) The PAM CRISPR/Cas12a-assisted RPA assay entails adding Cas12a, a CRISPR guide RNA (crRNA) that is immediately downstream to the protospacer adjacent motif (PAM) associated with the Cas12a, and a fluorogenic reporter that is terminally labeled with a fluorophore and a quencher. During the reaction, the presence of RPA amplicons leads to Cas12a activation and cleavage of the fluorogenic reporter, thus producing a fluorescence signal. (D) The PAM-free CRISPR/Cas12a-assisted RPA assay uses the same Cas12a and fluorogenic reporter as its PAM counterpart and fluorescently detects RPA amplicons similar to its PAM counterpart but uses a different crRNA that is without an upstream PAM. (E) Comparison of assay design, operation, signal, sensitivity, speed, and cost helps identify advantages and disadvantages of the CRISPR/Cas12a-assisted RPA assays with respect to the CRISPR/Cas12a-free assay, thereby pinpointing the role and impact of CRISPR/Cas12a in these assays.

Figure 2.

Assay optimizations for systematic comparisons between three RPA assays. (A) For the CRISPR/Cas12a-free RPA assay, combining (i) 100 nM exo probe and (ii) 150 nM RPA primers yields a strong normalized end-point fluorescence signal from 200 copies/μL (0.14) target without elevating the signal for the NTC (0.01). (B) For the PAM CRISPR/Cas12a-assisted RPA assay, combining (i) 320 nM RPA primers and (ii) 1280 nM crRNA–Cas12a results a strong end-point signal of 4.47 from 2000 copies/μL target, a detectable end-point signal of 0.30 from 20 copies/μL target, and a low end-point signal of 0.02 from the NTC. (C) For the PAM-free CRISPR/Cas12a-assisted RPA assay, combining (i) 320 nM RPA primers and (ii) 640 nM crRNA–Cas12a produces the strongest end-point signals of 13.35 and 1.10 from 2000 and 20 copies/μL target, respectively, while maintaining a low end-point signal of 0.07 from the NTC.

Figure 3.

Formulating mechanistic framework for PAM and PAM-free CRISPR/Cas12a-assisted RPA assays. (A) Gel electrophoresis of reaction products from PAM and PAM-free CRISPR/Cas12a-assisted RPAs shows clearer RPA amplicon and cis-cleaved fragment bands from the PAM-free CRISPR/Cas12a-assisted RPA than from its PAM counterpart, with the brightest bands with 640 nM PAM-free crRNA–Cas12a. (B) The PAM crRNA–Cas12a is preferentially activated by dsDNA activators than by ssDNA activators, whereas the PAM-free crRNA–Cas12a is preferentially activated by ssDNA activators than by dsDNA activators. (C) The crRNA–Cas12a complex in the PAM CRISPR/Cas12a-assisted RPA assay can recognize and cis-cleave double-stranded RPA amplicons, resulting in impediment of exponential amplification. Such impediment leads to fewer activated Cas12a and incomplete trans-cleavage of the fluorogenic reporter. For the PAM-free CRISPR/Cas12a-assisted RPA, the crRNA–Cas12a complex is preferentially accessible to its single-stranded complementary region that is temporarily available during DNA polymerization, leading to less impediment of exponential amplification and thus generates a stronger signal via stronger trans-cleavage activity than its PAM counterpart.

Figure 4.

Comparison of assay sensitivity, speed, and cost. For each assay, (i) sensitivity is evaluated by measuring the end-point signals from 2000, 200, 20, and 2 target copies/μL and the no target control (NTC) in triplicate (n = 3) and performing Student’s t-tests, (ii) speed is evaluated by measuring the “times-to-positive”—the time at which the signal from the target becomes detectable above the NTC—in triplicate (n = 3), and (iii) cost is evaluated by totaling the reagent cost of a 10 μL reaction at the fine-tuned reagent concentrations. (A) The CRISPR/Cas12a-free assay (i) produces low but detectable end-point signals from only 2000, 200, and 20 copies/μL (p < 0.05) while (ii) reporting fast times-to-positive and (iii) costing $0.87. (B) The PAM CRISPR/Cas12a-assisted assay (i) generates stronger end-point signals among 2000, 200, and 20 copies/μL (p < 0.05) with (ii) longer times-to-positive, and (iii) a nearly 3-fold higher cost of $2.54 due to its high concentration of crRNA–Cas12a. (C) The PAM-free CRISPR/Cas12a-assisted assay (i) yields strongest end-point signals from 2000, 200, and 20 copies/μL (p < 0.05) and can detect 2 copies/μL (signal =0.71 ± 0.07, n = 2) while (ii) posting the fastest times-to-positive, although (iii) its cost of $1.64 due to its reduced concentration of crRNA–Cas12a remains high. (D) The PAM-free CRISPR/Cas12a-assisted assay with an alternative Cas12a from Integrated DNA Technology (IDT) (i) produces weaker but detectable end-point signals from 2000, 200, and 20 copies/μL (p < 0.05) and also manages to detect 2 copies/μL (signal = 0.54 ± 0.09, n = 2). Despite (ii) longer times-to-positive, its (iii) cost is only $0.99 due to its low concentration of crRNA–Cas12a. Data presented are in (i) and (ii) as mean ± SD. Red dashed line represents signal thresholds at mean + 3SD of the NTCs.