Programmable RNA detection with CRISPR-Cas12a

Abstract

CRISPR is a prominent bioengineering tool and the type V CRISPR-associated protein complex, Cas12a, is widely used in diagnostic platforms due to its innate ability to cleave DNA substrates. Here we demonstrate that Cas12a can also be programmed to directly detect RNA substrates without the need for reverse transcription or strand displacement. We discovered that while the PAM-proximal "seed" region of the crRNA exclusively recognizes DNA for initiating trans- cleavage, the PAM-distal region or 3'-end of the crRNA can tolerate both RNA and DNA substrates. Utilizing this property, we developed a method named Split Activators for Highly Accessible RNA Analysis or 'SAHARA' to detect RNA sequences at the PAM-distal region of the crRNA by merely supplying a short ssDNA or a PAM containing dsDNA to the seed region. Notably, SAHARA is Mg ²⁺ concentration- and pH-dependent, and it was observed to work robustly at room temperature with multiple orthologs of Cas12a. SAHARA also displayed a significant improvement in the specificity for target recognition as compared to the wild-type CRISPR-Cas12a, at certain positions along the crRNA. By employing SAHARA we achieved amplification-free detection of picomolar concentrations of miRNA-155 and hepatitis C virus RNA. Finally, SAHARA can use a PAM-proximal DNA as a switch to control the trans-cleavage activity of Cas12a for the detection of both DNA and RNA targets. With this, multicomplex arrays can be made to detect distinct DNA and RNA targets with pooled crRNA/Cas12a complexes. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.

Fig. 1:. Cas12a orthologs tolerate short ssDNA activators (6–12 nt) when added in combination

a. Schematic representation of a crRNA-Cas12a complex performing trans-cleavage of ssDNA reporters following the recognition of two split-activators. b-d. Fold change at t=60 minutes of in vitro trans-cleavage assay with Cas12a orthologs (red = LbCas12a, green = AsCas12a, orange = ErCas12a) activated by individual truncated ssDNA activators of length 6–20 nt e-g. Heat maps representing fold change at t=60 minutes of an in vitro trans-cleavage assay activated by combinations of truncated ssDNA activators of different lengths ranging from 6–14 nt in the Pp and Pd regions. The reactions contained 25 nM truncated ssDNA GFP-activators, 60 nM Cas12a, and 120 nM crGFP and were incubated for 60 min at 37°C. Error bars represent SD (n=3). Statistical analysis was performed using a two-tailed t-test where ns = not significant with p > 0.05, and the asterisks (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001) denote significant differences.

Fig. 2. Split-activator detection of ssDNA, dsDNA, and RNA substrates by Cas12a:

a. Schematic representation of Cas12a activated by combinations of ssDNA (red), dsDNA (orange), and RNA (blue) in the PAM proximal and PAM distal regions. b-d Heat maps representing the fold changes of in vitro trans-cleavage assay (n=3) with Cas12a orthologs for the combinatorial schemes seen in (a). e-h Comparison of the WT crRNA and ENHANCE crRNA for in vitro trans-cleavage assay split activators. Note, ssDNA and ssRNA substrates were used as targets in the Pd region while dsDNA was supplied in the Pp. Reactions were incubated for 60 min at 37°C. Error bars represent SD (n=3). The reactions contained 25 nM of each truncated activator, 60 nM Cas12a, and 120 nM crRNA.

Fig. 3:. Development of SAHARA for the detection of a wide range of RNA targets

a. Schematic representation of Cas12a complexed with WT vs. SAHARA crRNA and activated by either a short (20-nt) or long (730-nt) RNA activators. b-d. Comparison of trans-cleavage activity among Cas12a orthologs for the short vs. long combinatorial schemes seen in (a). The plot represents raw fluorescence units (RFU) plotted for time t=60 min. e-g. Detection of RNA target with SAHARA by using either a non-targeting scrambled S12 (SR-Scr) or a targeting S12 (SR-S12) The plot represents RFU at t=60 min All error bars represent sd (n=3). Statistical analysis was performed using a two-tailed t-test where ns = not significant with p > 0.05, and the asterisks (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001) denote significant differences.

Fig. 4:. Application of SAHARA for the detection of HCV and miR-155

a. Schematic of an HCV polypeptide precursor RNA target and three crRNAs targeting it at different positions. Colors within the target and crRNA indicate the following: crRNA Pp region (green), head (orange), tail (purple), and middle (blue) sections of an HCV polypeptide precursor RNA. b. Comparison among the head, tail, mid, and pooled HCV targeting crRNA. The plot represents the fold change in fluorescence intensity normalized to the NTC at t=60 min (n=3). c. Limit of detection of HCV target using a pool of head, tail, and mid crRNA sequences. The plot represents the background-subtracted fluorescence intensity at t=60 min, for different concentrations of the target. d. Head vs Tail detection for a mature miRNA-155 target meditated by a split activator system. crRNAs were designed to target an S12 dsDNA GFP-activator in the Pp region and target either the head or tail region of a miR-155 target in the Pd region. e. Comparison of normalized fluorescence intensity fold change values among cr155-Tail, cr155-Head, and a combination of both Head and Tail targeting crRNAs. f. miR-155 limit of detection using a pooled crRNA with a split activator system. The plot represents the background-subtracted fluorescence intensity at t=60 min, for different concentations of the target. Error bars represent SD (n=3). Statistical analysis was performed using a two-tailed t-test where ns = not significant with p > 0.05, and the asterisks (*P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001) denote significant differences.

Fig. 5:. Specificity of SAHARA towards single point mutations in target

a. Schematic of WT vs SAHARA CRISPR-Cas systems for the detection of a target nucleic acid. b. ssDNA activators were designed with point mutations across the length of the activator. GFP-activator mutants were designed for a WT CRISPR activator (24-nt) and a SAHARA split activator system (12-nt +12-nt). The mutation location is identified by ‘M’ following the nucleotide number where the base has been changed to guanine (3’ to 5’ direction). c-e. Comparison of fold changes for the in vitro trans-cleavage assay between WT and SAHARA activator mutants normalized to the WT activator for Cas12a orthologs (c: LbCas12a, d: AsCas12a, and e: ErCas12a). Comparison of RFU values at t=60 min for the in vitro trans-cleavage assay between WT and SAHARA. Statistical analysis was performed using a two-tailed t-test where ns = not significant with P > 0.05, and the asterisks (*P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001) denote significant differences.

Fig. 6. Characterizing properties of the S12 DNA binding at the Pp region in SAHARA:

a-d. PAM sequence tolerance of Cas12a orthologs (red = LbCas12a, green = AsCas12a, orange = ErCas12a) coupled with SAHARA. Comparison of trans-cleavage activity among S12 dsDNA activators containing different PAM sequences (n=3). The PAM sequences TTTA, AAAT, and VVVN were assessed. e-g. Cas12a orthologs tolerate a wide range of GC contents in the crRNA and S12 dsDNA for RNA detection (n=3). h-j. The trans-cleavage activity of Cas12a with varying concentrations of S12 after incubation for 60 min at 37°C. Error bars represent SD (n=3).

Fig. 7. Simultaneous detection of multiple targets with SAHARA

a. Schematic of multiplexed detection with SAHARA. A mixture of different crRNAs can be differentiated for trans-cleavage activity by the use of sequence-specific S12 activators. b-d. Heat maps depicting the trans-cleavage activity of 3 different pooled crRNAs (crRNA-a, crRNA-b, and crRNA-c) in the presence of 3 different S12 activators (S12a, S12b, S12c) or a no S12 control for Lb, As, and Er cas12a orthologs. Fold change compared to the NTC at t=60 min from the start of the reaction is plotted. 30 nM Cas12a, 60 nM crRNA, 25 nM S12 activators, and 25 nM of DNA or RNA targets were used in the assay (n=3). e. Schematic of multiplexed RNA detection with a combination of SAHARA and Cas13b. DNA or RNA reporters consisting of different colored dyes are used to distinguish the signal produced by Cas12a and Cas13b. f-g. Multiplexed RNA detection using Lb, As, and Er orthologs of Cas12a and PsmCas13b. Cas12a targets activator T1 and produces a signal in the FAM channel, while Cas13b targets activator T2 and produces a signal in the HEX channel (n=3).