To precisely calculate the Cas12a trans-cleavage activity, the ssDNA reporter used was dual labeled with fluorophore and quencher (i.e., FQ-reporter), and the reaction was monitored by a fluorescence reader. To simplify the calculation, the trans-cleavage activity was calculated on the basis of the initial fluorescence growth rate v_g (v_g = ΔF/Δt), following the recently published Michaelis-Menten model (Ramachandran and Santiago, 2021).

Non-paired ssDNA FQ-reporters are the substrates for Cas12a trans-cleavage reactions, and the sequences may affect the cleavage efficiencies (Li et al., 2018a). We first tested four homopolymers of 6 nucleotides in length, including poly-adenine (A), poly-cytosine (C), poly-guanine (G), and poly-thymine (T), respectively, and found Cas12a trans-cleaved the poly-C reporter with the highest efficiency but failed to cleave the poly-G reporter (Figures 1A,B), which was consistent with the previous findings (Yue et al., 2021). Along with the elongation of the ssDNA reporter sequence, the v_g gradually increased and reached the peak when the sequence was longer than 8 nucleotides (Figures 1C,D). Considering the fact that longer ssDNA FQ-reporter has higher background fluorescence and is more expensive to synthesize, the 8C FQ-reporter was chosen for the following assays.

To optimize the Cas12a reaction buffer, we first compared several commercially available reaction buffers and found that Cas12a in buffer F had the highest trans-cleavage efficiencies (Figure 2A and Supplementary Table 1). As the pH values may have influences on the Cas12a trans-cleavage activities, a group of commercially available standard buffers with pH values ranging from 3.5 to 9.6 in increments of 0.1 were further analyzed, through individually substituting the (hydroxymethyl)aminomethane (abbreviated as Tris) salt in buffer F. Among the tested conditions, Cas12a generally preferred buffers with high pH values and exhibited obviously higher trans-cleavage activities in the 1 M glycine buffer (pH 8.6) and the 1 M Tris–HCl buffer (pH 8.5) (Figure 2B). Considering the buffering capacity in maintaining a stable pH value of the reaction buffers, especially when complicated samples are detected, the Tris–HCl buffer (pH 8.5) was finally chosen. Meanwhile, the loss of Cas12a trans-cleavage activities in buffer C could be caused by either the relatively low pH value or the low concentration of Mg²⁺, which has been demonstrated to be important for the maintenance of Cas12a cis-cleavage activities in a previous study (Fonfara et al., 2016; Supplementary Table 1).

Meanwhile, dozens of additives were individually added into the reaction buffer F to test their efficacy on the Cas12a trans-cleavage activities, and several components such as polyethylene glycol (PEG) and glycerol obviously enhanced the cleavage signals and were chosen for further analysis (Figure 2C). The chosen additives of different combinations and concentrations were compared and the condition of 0.4% PEG-20000 showed the most remarkable enhancement and was finally selected (Supplementary Figure 1). In addition, we found that non-ionic detergents such as Triton X-100 could also promoted the Cas12a trans activities.

After systematical analysis of the above selected components, an optimized reaction buffer namely HOLMES Buffer 1 showed better performance than buffer F in triggering the Cas12a trans-cleavage and was chosen for subsequent analysis (Supplementary Figure 2 and section “5.3.1” in Supplementary Protocol). Moreover, through comparison with the control buffer of NEB buffer 3.1, HOLMES Buffer 1 remarkably enhanced the Cas12a trans-cleavage activities as well as its detection sensitivities (Figure 2D).

Besides the reaction buffer, the ratio among Cas12a, crRNA, and target dsDNA could also be an important factor affecting the Cas12a trans-cleavage efficiencies, and different combinations were therefore tested in HOLMES Buffer 1. In theory, excessive amounts of crRNA and target dsDNA could promote the formation of the Cas12a ternary complex as well as triggering of the Cas12a trans-cleavage; however, we found that the Cas12a cleavage activity was slightly inhibited by high concentrations of crRNA and dsDNA with an unknown reason (Supplementary Figure 3). Therefore, a ratio of 1: 2: 2 was finally chosen to obtain the best kinetic parameters for Cas12a trans-cleavage in HOLMES Buffer 1.

At the presence of the target DNA, the Cas12a trans-cleaves the ssDNA FQ-reporter to illuminate fluorescent signals, which can be employed to measure the Cas12a enzymatic constants. However, as the fluorescence intensities may vary from different readers, standard curves should be first created to correlate the relative fluorescent units (RFUs) and the concentrations of the FQ-reporter, including both un-cleaved (i.e., the substrates) and the cleaved (i.e., the products) reporters (Figures 3A,B and Supplementary Figures 4a,b). Briefly, the ssDNA FQ-reporter should be serially diluted and both cleaved and un-cleaved fluorescence signals are continually measured till the fluorescence reaches the saturation point, and the highest values of each gradient are then used for calibration of the standard curves for both cleaved and un-cleaved reporters (Figures 3C,D and Supplementary Figures 4c,d).

Then, with the employment of a limited concentration of Cas12a and crRNA and varied concentrations of ssDNA reporters, trans-cleavage enzymatic kinetics for Cas12a were determined, including the values of v_max, K_m, and K_cat (Figure 4 and Supplementary Table 2). Noticeably, the constants of K_m, K_cat as well as the K_cat/K_m were miscalculated in a previous study (Chen et al., 2018), which were much higher than the data in this study and another work (Ramachandran and Santiago, 2021) and have been recently corrected (Supplementary Table 3).

In the above-mentioned optimized reaction condition, the Cas12a trans-unit (transU) was defined as the amount of Cas12a that trans-cleaves 1 pmol 8C FQ-reporter in 1 min at 37°C in a total reaction volume of 20 μL in HOLMES Buffer 1. To simplify the transU measurement, a step-by-step protocol was provided in the Supplementary Protocol, which can be run on different fluorescence readers. Noticeably, as the Cas12a trans-cleavage efficiencies can be remarkably affected by the crRNA and target dsDNA sequences as well as their concentrations, the transU should be determined using the same sequences and concentrations as provided in this study to unify the measurement (ref to sections “5 and 6.2” in Supplementary Protocol).

Briefly, a standard curve correlating the RFUs and the reporter concentrations should be drawn first, and then the trans-cleavage reaction is performed with serially diluted Cas12a and excess ssDNA reporters. The obtained data in the linear region are then used for calculation of the Cas12a transU. Following this protocol, a commercially available LbCas12a was then analyzed, and the trans-cleavage activities were determined to be 51.65 transUs/pmol (i.e., 19.36 fmol per transU) (Figure 5).

Taken together, we here systematically optimized the Cas12a trans-cleavage reaction conditions, measured the Cas12a enzymatic constants and defined its transU, which will undoubtedly facilitate the development and application of robust Cas12a-based CRISPR-Dx systems in the future. Moreover, following a similar approach, the transU of other Cas effectors such as Cas12b, Cas12f, and Cas13 can be defined and their measurement protocols can be easily developed. On the other hand, although Cas12a exhibits much better performance in HOLMES Buffer 1 (e.g., with a higher K_cat value), one should be aware that there is still much work to do to further improve the Cas12a trans-cleavage activities, which may include the optimization of not only the reaction buffers but also the crRNA sequences in the future.

LbCas12a (LbCpf1) (Cat. No. #32108-03) was ordered from Tolo Biotech. (Shanghai, China). crRNAs were chemically synthesized by Biolino Acid Technology (Tianjin, China). FQ-labeled ssDNA reporters were ordered from Sangon Biotech. (Shanghai, China). The additive screening plates (Cat. No. #HR2-072) and the pH slice plate (Cat. No. #HR2-070) were ordered from Hampton Research (Aliso Viejo, CA).

For the short dsDNA target, two complementary oligonucleotides were synthesized by commercial companies and then annealed to form dsDNA. Specifically, a pair of oligonucleotides (4 μM each) were mixed in 1× PCR buffer and then annealed in the following procedure with a thermal cycler: 95°C for 5 min and then reduced from 95 to 20°C at a rate of 1°C per minute. The annealed dsDNA target was diluted to 1 μM and stored at −20°C before use.

The Cas12a trans-cleavage reaction was performed in a 20-μL volume, following the conditions as described before (Li et al., 2018b). In detail, 50 nM crRNA, 5 nM Cas12a nuclease, 4 nM dsDNA target, and 200 nM 8C FQ-reporter were pre-mixed in 1× buffer F and then 16 μL of the mixture was aliquoted into the 96-well PCR plate, which should be operated on ice. Then, the reaction was initiated by adding 4-μL additives into each well and the fluorescence signals were measured by the qPCR machine (Applied Biosystems QuantStudio 3). The program was set at 37°C and signals (λex: 488 nm; λem: 535 nm) were collected every 15 s. The initial fluorescence growth rate (v_g) was calculated according to the maximum slope of the fluorescence curve using the ICEKAT software¹.

The Michaelis-Menten was analyzed according to following equation: v₀ = v_max [S]/(K_m + [S]), where [S] is the substrate concentration, v_max is the maximum reaction rate, and K_m is the Michaelis constant. The turnover number (K_cat) was determined by the equation: K_cat = v_max/[Et], where Et was the effective complex. The concentration of the activated Cas12a enzymatic complex was always kept at 1 nM in 1× reaction buffer. The Cas12a trans-cleavage reaction was initiated by the addition of the ssDNA FQ-reporter at the final concentrations of 0 nM, 3.9 nM, 7.8 nM, 15.6 nM, 31.2 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, and 1 μM, respectively. Reactions were carried out in three replicates at 37°C and fluorescence readouts were taken every 15 s. Background-subtracted fluorescence signals were obtained by subtracting the signal from a negative control. The v₀ data were converted to nM/s from Relative Fluorescent Units using the calibrated standard curve (ref to section “6.1” in Supplementary Protocol), and the Michaelis-Menten constants were determined by plotting v₀ against the substrate concentration and then fitting into the Michaelis-Menten curve (Graphpad Software).