TracrRNA reprogramming enables direct PAM-independent detection of RNA with diverse DNA-targeting Cas12 nucleases

Abstract

Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA's presence to Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram tracrRNAs from diverse Cas12 nucleases, linking the presence of an RNA-of-interest to dsDNA cleavage and subsequent collateral single-stranded DNA cleavage-all without the RNA necessarily encoding a protospacer-adjacent motif (PAM). After elucidating nuclease-specific design rules, we demonstrate PAM-independent RNA detection with Cas12b, Cas12e, and Cas12f nucleases. Furthermore, rationally truncating the dsDNA target boosts collateral cleavage activity, while the absence of a gRNA reduces background collateral activity and enhances sensitivity. Finally, we apply this platform to detect 16 S rRNA sequences from five different bacterial pathogens using a universal reprogrammed tracrRNA. These findings extend tracrRNA reprogramming to diverse dsDNA-targeting Cas12 nucleases, expanding the flexibility and versatility of CRISPR-based RNA detection.

Fig. 1. A framework for PAM-independent RNA detection by tracrRNA-dependent Cas12 nucleases with PUMA.

a Overview of Cas12 Rptr-based RNA detection platform. Top: Traditional Cas12-based diagnostics use gRNAs to recognize and cut dsDNA targets, triggering trans-cleavage activity for signal visualization and amplification. However, selected targets must be flanked by a PAM sequence and cannot directly detect RNA. Bottom, Reprogrammed tracrRNAs (Rptrs), binding and converting sensed RNAs into guide RNAs, enable direct and PAM-independent RNA detection. b Known tracrRNA-encoding CRISPR-Cas systems. The tracrRNA is encoded in all Type II subtypes and in 8 of 14 defined Type V subtypes, including V-B, -C, -D, -E, -F, -G, -K, and -L. The CRISPR locus architecture is based on prior work^,,. c Representative examples of five different duplexes formed between the tracrRNA and crRNA among tracrRNA-encoding CRISPR-Cas systems. In type II CRISPR-Cas systems, a single imperfect RNA duplex is formed between the crRNA repeat and tracrRNA anti-repeat (R/AR). In contrast, the V-B, -F, and -G systems form two duplexes (long repeat/anti-repeat duplex, LR/AR; short repeat/anti-repeat duplex, SR/AR), the V-E and -K systems form one LR/AR duplex and one triple helix, and the V-C system forms three R/AR duplexes. PmuCas12c1, Cas12c from Parasutterella muris, PDB: 7VYX. See detailed information in Supplementary Fig. 1.

Fig. 2. TracrRNA reprogramming enables sequence-specific RNA detection by Cas12b.

a Predicted tracrRNA-crRNA structure for BhCas12b based on its ortholog BthCas12b (PDB: 5WTI). R/AR, repeat/anti-repeat. b Setup to assess Rptr functionality using cell-free transcription-translation (TXTL). Expressed Cas-guide RNA complex recognizes and cuts its dsDNA target, causing the degradation of target-encoding GFP reporter plasmid and resulting in lower fluorescence compared to a non-targeting guide control. c 16-hour endpoint fluorescence measurements in TXTL when changing the long and short RNA duplexes. NT, non-targeting guide; T, targeting guide; T-br, targeting crRNA with bulge removed. d Setup to reprogram tracrRNAs to sense a Campylobactor jejuni transcript CJ8421_04975 mRNA. The guide and target components are added in the form of DNA constructs, while the purified BhCas12b protein is used. mRNA(mut), mRNA with point mutations in the predicted seed region of the guide. Rptr(scr-LA), Rptr with the long anti-repeat sequence scrambled; Rptr(scr-SA), Rptr with the short anti-repeat sequence scrambled; Rptr(scr-LA&SA), Rptr with both long and short anti-repeat sequence scrambled. e 16-hour endpoint fluorescence measurements in TXTL when assessing Rptr-guided sequence-specific dsDNA targeting. Nucleotide changes in R/AR stems in c and d are indicated by gray boxes. Bars and error bars in c, e represent the mean and standard deviation from three independently mixed TXTL reactions. Dots represent individual measurements. ***p < 0.001 based on a one-sided Student’s t-test with unequal variance (n = 3). Source data are provided as a Source Data file.

Fig. 3. TracrRNA reprogramming enables sequence-specific RNA detection by Cas12f1 and Cas12e.

a AsCas12f1 sgRNA structure (PDB: 8J12). See the detailed information in Supplementary Fig. 1. b 16-hour endpoint fluorescence measurements in TXTL when reprogramming the long and short RNA duplexes in the AsCas12f1 sgRNA. NT, non-targeting crRNA; T, targeting crRNA. c Setup to detect the Campylobacter jejuni transcript CJ8421_04975 mRNA using AsCas12f1 Rptrs in TXTL. d 16-hour endpoint fluorescence measurements in TXTL for Rptr-guided sequence-specific dsDNA targeting by AsCas12f1 in TXTL. e Structure of DpbCas12e sgRNA (PDB: 6NY3). In the triple-helix region, a cis Hoogsteen/Watson-Crick base pair is formed between the U.A and a cis Watson-Crick/Watson-Crick base pair between the A-U. f 16-hour endpoint fluorescence measurements in TXTL when assessing the changeability of the LR/AR region. Dpb_T-br, targeting sgRNA with the bulge and G.U wobble base pair removed. g 16-hour endpoint fluorescence measurements in TXTL when changing the RNA triple-helix region. h 16-hour endpoint fluorescence measurements in TXTL when changing the RNA triple-helix surrounding region. i, Setup to detect the Campylobacter jejuni CJ8421_04975 mRNA using DpbCas12e Rptrs in TXTL. j, 16-hour endpoint fluorescence measurements for Rptr-guided sequence-specific dsDNA targeting by DpbCas12e in TXTL. Rptr(scr-dplx), Rptr with a scrambled anti-repeat sequence; Rptr(scr-tplx), Rptr with the RNA triple-helix sequence scrambled; Rptr(scr-d&tplx), Rptr with the RNA duplex and triple-helix sequence scrambled. Nucleotide changes in AsCas12f1 sgRNA and DpbCas12e sgRNA in b, f, g and h are indicated by gray boxes. Bars and error bars in b, d, f, g, h, and j represent the mean and standard deviation from three independently mixed TXTL reactions. Dots represent individual measurements. No error bars are shown when only two replicates were successfully collected. *: p < 0.05. **: p < 0.01. ***:p < 0.001 based on a one-sided Student’s t-test with unequal variance (n = 3). Source data are provided as a Source Data file.

Fig. 4. Truncating dsDNA targets enhances collateral cleavage activity.

a Schematic of the in vitro trans-cleavage assay. The assay includes purified a Cas12 nuclease, an in vitro transcribed Rptr, and a linear dsDNA target. The Cas12-guide RNA ribonucleoprotein (RNP) recognizes and cleaves its dsDNA target, which triggers non-specific cleavage activity on ssDNA. Specifically, cleavage of the non-target strand (NTS) occurs before cleavage of the target strand (TS). F, fluorophore; Q, quencher. Yellow circle, PAM; b Impact of unprocessed or processed targets on in vitro trans-cleavage activity by BhCas12b. TS cleavage is the rate-limiting step. Red arrow, cleavage site. The cleavage site of TS is set as position 0. -, truncating the target sequence on NTS or TS. +, adding an overhang on NTS or TS. The PAM is in brown and the target is in blue. c, Direct detection of the full-length CJ8421_04975 mRNA by BhCas12b based on in vitro collateral cleavage activity. Yeast RNA is added in the same mass amount as the 1000 nM sensed mRNA, and the best-performing dsDNA target NTS-6: TS-2 is used. d, Impact of unprocessed or processed targets on in vitro collateral cleavage activity by DpbCas12e. e Direct detection of the full-length CJ8421_04975 mRNA by DpbCas12e based on in vitro collateral cleavage activity. Yeast RNA is added in the same mass amount as the 1000 nM sensed mRNA, and the best-performed dsDNA target NTS-8: TS-4 is used. 16 h end-point values were used to make the plots in c and e. See Supplementary Figs. 6b and 10a for the complete time courses. Curves in b and d represent the mean from two independent collateral assays. Bars and dots in c and e represent the mean and individual measurements, respectively, from two independent collateral cleavage assays. Light blue bars indicate the limit-of-detection (LOD) conservatively estimated as the lowest concentration yielding an average fluorescence exceeding 50% of that of the no-RNA control. Source data are provided as a Source Data file.

Fig. 5. PUMA exhibits reduced background collateral activity due to the absence of a traditional single-guide RNA.

a, Measured in vitro collateral cleavage activity with BhCas12b and an sgRNA with or without a dsDNA target. b, Measured in vitro collateral cleavage activity with BhCas12b and a Rptr and a dsDNA target with or without the sensed RNA. c, Sensitivity comparison between sgRNA and Rptr. In a-b, 37-bp NTS-2:TS-2 processed dsDNA targets were used for both sgRNA and Rptr. In c, 334-bp DNA fragments containing the core PAM-flanking target were used with the sgRNAs and 37-bp NTS-2:TS-2 processed dsDNA targets were used with the Rptrs. In a-c, sgRNA#1 and sgRNA#4 share the same guide sequences as those generated by Rptr#1 and Rptr#4, respectively. Dots represent individual measurements from two independent collateral cleavage assays. Bars represent the mean of the dots. In a-b, values represent fluorescence measurements after reaction times of 2 hours and 16 hours. In c, values represent fluorescence measurements after reaction times of 16 hours. Light blue bars indicate the limit-of-detection (LOD) conservatively estimated as the lowest concentration yielding an average fluorescence exceeding 50% of that of the no-RNA control. Source data are provided as a Source Data file.

Fig. 6. A universal Rptr enables sequence-specific detection of 16 S rRNA from different bacterial pathogens.

a Tolerance of guide-target mismatches for three different Cas12b orthologs based on in vitro collateral cleavage activity. The DNA target is the same as the one used in Fig. 4B (BhsgRNA4 DNA target). Heat maps represent the mean kobs values from two independent collateral assays. See the k_obs values in Supplementary Data 1. b Setup to differentiate 16 S rRNA from five different pathogens using only one universal BthCas12b Rptr binding to a conserved region of 16 S rRNA. A truncated long anti-repeat of 18 nts instead of the usual 31 nts is used in the universal Rptr. In the alignment, sequences that match the E. coli 16 S rRNA are in black, while those that do not match are shown in red. c Detection of pathogen 16 S rRNAs with a universal Rptr and corresponding dsDNA targets based on in vitro collateral cleavage activity with BthCas12b. Partial 16 S rRNA fragments of different pathogens at a final concentration of 100 nM were used. Values represent 36-minute reaction times. Values in c represent the mean and standard deviation from two independent collateral assays. Source data are provided as a Source Data file.