RAPID: Evaluation of Cas12a Protospacer Nicking and Chimeric Reporters for PAM-free RNA and DNA diagnostics

RAPID: Evaluation of Cas12a Protospacer Nicking and Chimeric Reporters for PAM-free RNA and DNA diagnostics | medRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-P4HH5NV'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search RAPID: Evaluation of Cas12a Protospacer Nicking and Chimeric Reporters for PAM-free RNA and DNA diagnostics View ORCID Profile Idorenyin A. Iwe , Frank X. Liu , Ariel Corsano , Severino Jefferson Ribeiro da Silva , Jennifer Doucet , View ORCID Profile Serena Singh , View ORCID Profile Gabriel Lamothe , Riham Zayani , Jessica Nguyen , Quinn Matthews , Justin RJ. Vigar , Pouriya Bayat , Mohammad Simchi , Kristof Bozovicar , Moiz Charania , Sabina Panfilov , XiuJun Li , Tony Mazzulli , Jacques P. Tremblay , Yufeng Zhao , View ORCID Profile Alexander A. Green , Zhigang Li , View ORCID Profile Shuhuai Yao , View ORCID Profile Keith Pardee doi: https://doi.org/10.1101/2025.07.12.25331452 Idorenyin A. Iwe 1 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, ON M5S 3M2, Canada 2 Department of Mechanical and Industrial Engineering, University of Toronto , Toronto, ON M5S 1A1, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Idorenyin A. Iwe Frank X. Liu 3 Department of Mechanical and Aerospace Engineering, The Hong Kong University of Science and Technology , Hong Kong 4 Orange Biotech Limited , Hong Kong Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ariel Corsano 1 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, ON M5S 3M2, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Severino Jefferson Ribeiro da Silva 1 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, ON M5S 3M2, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jennifer Doucet 1 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, ON M5S 3M2, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Serena Singh 1 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, ON M5S 3M2, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Serena Singh Gabriel Lamothe 5 Department of Molecular Medicine, Faculty of Medicine, Université Laval , Québec, G1V 4G2 Québec, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gabriel Lamothe Riham Zayani 1 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, ON M5S 3M2, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jessica Nguyen 1 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, ON M5S 3M2, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Quinn Matthews 1 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, ON M5S 3M2, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Justin RJ. Vigar 1 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, ON M5S 3M2, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pouriya Bayat 1 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, ON M5S 3M2, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mohammad Simchi 1 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, ON M5S 3M2, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kristof Bozovicar 1 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, ON M5S 3M2, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Moiz Charania 1 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, ON M5S 3M2, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sabina Panfilov 1 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, ON M5S 3M2, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site XiuJun Li 6 Department of Chemistry & Biochemistry, University of Texas at El Paso , USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tony Mazzulli 7 Department of Laboratory Medicine and Pathobiology, University of Toronto , Toronto, M5S 1A8 ON, Canada 8 Department of Microbiology, Sinai Health System/University Health Network , Toronto, M5G 1X5 ON, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jacques P. Tremblay 5 Department of Molecular Medicine, Faculty of Medicine, Université Laval , Québec, G1V 4G2 Québec, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yufeng Zhao 1 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, ON M5S 3M2, Canada 9 Centre for Research and Applications in Fluidic Technologies, National Research Council Canada , Toronto, M5S 3G8 ON, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alexander A. Green 10 Department of Biomedical Engineering, Boston University , Boston, MA 02215, USA 11 Molecular Biology, Cell Biology & Biochemistry Program, Graduate School of Arts and Sciences, Boston University , Boston, MA 02215, USA 12 Biological Design Center, Boston University , Boston, MA 02215, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alexander A. Green Zhigang Li 3 Department of Mechanical and Aerospace Engineering, The Hong Kong University of Science and Technology , Hong Kong Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: keith.pardee{at}utoronto.ca mezli{at}ust.hk meshyao{at}ust.hk Shuhuai Yao 3 Department of Mechanical and Aerospace Engineering, The Hong Kong University of Science and Technology , Hong Kong Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Shuhuai Yao For correspondence: keith.pardee{at}utoronto.ca mezli{at}ust.hk meshyao{at}ust.hk Keith Pardee 1 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, ON M5S 3M2, Canada 2 Department of Mechanical and Industrial Engineering, University of Toronto , Toronto, ON M5S 1A1, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Keith Pardee For correspondence: keith.pardee{at}utoronto.ca mezli{at}ust.hk meshyao{at}ust.hk Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract CRISPR-Cas nucleases have revolutionized diagnostics and biotechnology by providing programmable specificity. Here, we extend the understanding of Cas12a biology with a screen that, unexpectedly, finds that Cas12a trans cleavage activity can be modulated by nicks in the protospacer in a position-dependent manner. Wanting to explore the impact of non-conventional trans cleavage substrates, we subsequently find that non-specific Cas12a cleavage can be significantly reduced with RNA and chimeric (mixed RNA/DNA) reporter sequences. Exploiting these features, we introduce RAPID ( R NA/DNA A dvanced chimeric, P AM-free, I ntegrated Nicking, D iagnostics), a PAM-independent nucleic acid detection platform. By strategically introducing a nick within the spacer region, RAPID expands Cas12a detection to include target RNAs, which can be ligated in situ to create a hybrid protospacer-target with trans cleavage activity matching conventional Cas12a. We then apply RAPID to detect single point mutations in ssDNA and RNA substrates, a challenge for traditional Cas12 and Cas13 systems. In combination with RT-LAMP, RAPID is used for PAM-free RNA detection in clinical samples, achieving sensitivity down to ∼1 aM and 100% concordance with RT-qPCR. INTRODUCTION CRISPR-Cas12a (Cpf1), a class 2, type V endonuclease, is an RNA-guided nuclease that performs cis -cleavage of dsDNA (or ssDNA) targets and indiscriminate trans cleavage of ssDNA ( 1 , 2 ). The programmable and specific targeting capabilities of Cas12a have made it a widely applied tool in both nucleic acid detection and gene editing ( 3 – 5 ). Cas12a-based diagnostics often also incorporate an isothermal amplification method, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), to enhance detection sensitivity ( 5 – 7 ). Amplification is necessary due to the inherent sensitivity limitations of Cas12a, which typically operates down to the picomolar level ( 8 ). Combined with isothermal pre-amplification, the genetic material of viral pathogens (e.g. human papillomavirus (HPV), Ebola, Zika, and SARS-CoV-2) has been previously detected with sensitivity in the attomolar range, which is comparable to PCR ( 7 , 9 – 13 ). CRISPR methods thus provide next-generation diagnostic approaches for low-burden clinical-grade point-of-need testing without complex instrumentation ( 14 ). Recent advances in CRISPR-based diagnostics have focused on improved enzyme variants and gRNA (or CRISPR RNA) design to enable multimodal nucleic acid diagnostics (e.g. either DNA or RNA) with enhanced sensitivity, rapidity, specificity, and the capacity for multiplexed testing. More specifically, the newly characterized Cas12a2 and Cas12g variants exclusively target RNA substrates, which broadens the potential applications of the Cas12 family compared to DNA targeting alone ( 15 , 16 ). Moreover, gRNA alterations, including 5’ extensions and sequence splits have improved the kinetics of the CRISPR system as well as introduced multiplexing capabilities. ( 17 , 18 ) As tandem gRNA can increase sensitivity in both Cas12 and Cas13 systems ( 19 , 20 ), an engineered enzyme containing two gRNA-integrated regions has also enhanced the limit of detection ( 21 , 22 ). Protospacer engineering is another strategy to broaden Cas12a’s diagnostic capabilities, as demonstrated by split activators for DNA and RNA detection ( 23 , 24 ). Here, we focus on engineering the structure and composition of the nucleic acids in complex with the Cas12a enzyme. To date, there has been limited exploration of protospacer nicks that compromise activator integrity, or the composition of trans cleavage materials. Moreover, the recovery of Cas activation signal in the presence of pre-existing nicks in the DNA substrate remains unexplored. To unlock Cas12a’s full potential for nucleic acid targeting, the requirement for a protospacer adjacent motif (PAM) in target sequences must be alleviated. Engineering Cas9 has yielded variants that possess relaxed PAM requirements; meanwhile, introduction of PAM-presenting oligonucleotides (PAMmers) that are complementary to ssRNA targets has enabled PAM-free activation and cleavage by Cas9 ( 25 ). Thus far, PAM-free detection by Cas12a has only been achieved using ssDNA substrates, or methods based on toehold activation and temperature modulation ( 26 , 27 ). Advances in CRISPR-based diagnostics have also improved specificity towards detecting single point mutations ( 27 , 28 ), which can enable specificity for pathogen variants (e.g. influenza subtypes) or the detection of drug resistance, for example ( 29 ). However, to date, this requires additional sample preparation steps or high temperatures to convert dsDNA to ssDNA ( 20 , 26 ), features that are incompatible with high-throughput and field-based biosensing applications. An improved ability of Cas proteins to detect SNPs directly would significantly expand the range of diagnostic applications. Here, we begin with a fundamental screen that explores the effect of adding a nick in a stepwise manner across the protospacer sequence. Unexpectedly, we find that nicking modulates the activation of the Cas12a enzyme in a position-dependent manner, resulting in tunable nuclease activity, including muted, semi-muted, and complete activation. This phenomenon reveals that the Cas12a ribonucleoprotein’s ability to cleave nucleic acid targets relies not only on a PAM and base pairing, but also on the integrity of the target sequence at key loci. We also demonstrate, for the first time, that in situ ligation of such nicks can fully restore the Cas activation signal. Leveraging these findings, we have developed a PAM-independent nucleic acid targeting platform that primes Cas12a for activation using a separate PAM-duplex strand, independent of the target sequence. The result is unrestricted Cas12a targeting of sequences (e.g. no PAM constraint) and expands target substrates to include RNAs ( Fig. 1A ). Download figure Open in new tab Fig. 1: Tunable activation of CRISPR-LbCas12a RAPID system through target sequence breaks (A) Comparison of traditional CRISPR-Cas12a family targeting different nucleic acid substrates with the RAPID system. The RAPID system uniquely detects dsDNA, ssDNA, and RNA without requiring a PAM site on the target nucleic acid. (B) Demonstration of tunable activation of the RAPID system using LbCas12a, based on the nick location within the dsDNA. ( Methods 1-3 ). Complete activation is observed at positions P2 and P20, partial activation at P9 and P19, and no activation at P12. (C) Six foundational configurations of the RAPID system: (Type I) Configuration with continuous elongation of the target strand towards the PAM-distal region, while the non-target strand remains fixed. (Type II) Similar to (I), but with elongation extending into the PAM motif. (Types III & IV) Configurations analogous to (I) and (II), respectively, but without a non-target strand. (Type V) Utilizes ssDNA with strategic nick placements within the protospacer region. (Type VI) Features dsDNA with single-stranded breaks on the target strand. (D) Heat map showing normalized fluorescence intensities resulting from Cas12a activation and trans cleavage of the reporter for the six configurations described in (C), across 24 nick locations (P1 to P24) within the protospacer region, including the PAM. n=4 technical replicates; bars represent the arithmetic mean ± SD. To enhance the performance of the PAM-duplex, we next screened the effect of trans cleavage reporter composition on activity. Here, we introduce RNA homopolymers and chimeric (mixed RNA-DNA) reporter sequences. Through this approach, we discovered that Cas12a collateral cleavage reporting using modified RNA, rather than conventional ssDNA, significantly improves the signal-to-noise ratio. By combining the PAM-duplex with these new reporters, we developed a novel platform termed R NA/DNA A dvanced Chimeric, P AM-free, I ntegrated Nicking, D iagnostics (RAPID). This platform facilitates direct targeting of miRNA, RNA, ssDNA, and dsDNA downstream of the nick site without compromising Cas12a trans cleavage activity. RAPID also demonstrates enhanced performance when used with chimeric reporters. As a proof of concept, we then apply RAPID to detect point mutations in both ssDNA and miRNA, demonstrating improved specificity over conventional Cas12a detection ( 2 , 30 ). Interestingly, when combined with in situ ligation for the direct detection of RNAs, the result is a DNA-RNA hybrid template formed within the Cas12a clamp. Finally, with implementation of an upstream RT-LAMP (reverse transcription-loop-mediated isothermal amplification) reaction to boost target sequence concentration into the detectable range, we show that RAPID can identify mRNA in the attomolar range. Validation of Cas12a-based RAPID on SARS-CoV-2 clinical samples (Ct ≤ 33, 21 patient samples), targeting a PAM-free RNA target sequence, shows 100% concordance to the diagnostic performance of gold standard RT-qPCR assays. Taken together, we report new insights into the molecular regulation underpinning Cas12a trans cleavage through substrate nicking and chimeric reporters, and integrate these features into RAPID to provide high sensitivity, PAM-free detection, significantly expanding the CRISPR toolbox. RESULTS Platform development RAPID has been developed as a PAM-free CRISPR-Cas12 technology that extends detection to both DNA and RNA, something that has not been possible with conventional Cas12 technologies ( Fig. 1A ). We started our study with a systematic investigation of the relationship between single-stranded breaks across the CRISPR-Cas12a target sequence and enzymatic trans cleavage activation. Conventionally, in the presence of the target sequence, Cas12a introduces a staggered dsDNA break on the dsDNA target ( supplementary Fig. S1A), which subsequently triggers trans cleavage. Here, wanting to evaluate the importance of this dsDNA integrity on Cas12a function, we rationally introduced single-stranded breaks throughout the protospacer region of the target strand ( Fig. 1B , pink triangle), ( supplementary Fig. S1B) ( 31 , 32 ). Focusing on the sequence that interacts with the gRNA, we introduce single-stranded breaks in 24 positions starting from site between the first two PAM bases, denoted as P1, to the gRNA terminus adjacent nick, P24. ( Method 1&2 ). We then tested the effect of breaks at each site on Cas12a activation by monitoring trans cleavage of a standard quencher-/fluorophore-labelled ssDNA. The results were striking, with single stranded breaks having a clear position-dependent effect on activation ( Fig. 1B and supplementary Fig. S1C). The nuclease activity at P12 was entirely muted, with flanking regions exhibiting partial activation (semi-muted; P9, P19) and distal single stranded breaks (P2, P20) showing no inhibition. Having identified this unexpected impact on Cas12a activation, we then performed a comprehensive experiment with six different system configurations, designated as Types I through VI, each designed to interrogate specific aspects of Cas12a’s interaction with structurally diverse ssDNA and dsDNA substrates ( Fig. 1C ). The Type I configuration consists of dsDNA, wherein the non-target strand is unperturbed, and the target strand is incrementally extended from P1 to P24 to fulfill pairing interactions between the target strand and gRNA. Our objective was to identify the minimal target strand length necessary to trigger Cas12a activation, revealing that activation commenced at P18 on the target strand for Type I ( Fig. 1D ). Conversely, in Type II, the target strand was elongated from the distal end of the PAM region (P24) towards the PAM site (P1), with activation detected only when the target strand is extended to P6 or further. Types III and IV mimic the configurations of Types I and II, respectively, but with the non-target strand omitted to assess the influence of ssDNA on Cas12a activation. Our findings showed activation when the target strand is extended beyond P21 for Type III and when the target strand is extended from P24 to P7 or further for Type IV, generally reflecting the activation patterns of their Type I and II counterparts. Types V and VI systems display nicked ssDNA and dsDNA configurations, respectively. Type VI, provided a higher resolution data of the data previously discussed ( Figs. 1B and 1D), while Type V is comprised solely of a ssDNA target strand, featuring single strand breaks across positions P1 to P24. In Type V, Cas12a activation was either completely or partially impaired across several segments, notably from P7 to P15 and P19 to P20, when compared to Type VI. Taken together, these patterns of deactivation, which we found to correspond to regions that are known to interact with the endonuclease domain of Cas12a ( 33 ), lends support to our hypothesis that the presence of single strand breaks perturbs protein-DNA interaction dynamics. The activation patterns across these configurations are reported in the heatmap of Fig. 1D and the corresponding nucleic acid design sequences are presented in Table S1 . With the goal of fully understanding the Type VI design, we next compared the effect of introducing single stranded breaks into the target ( supplementary Fig. S1D, Method 3 )) and non-target ( supplementary Fig. S1E) strands. As expected, breaks in the target strand impair trans cleavage activation; however, interestingly, nicks in the non-target strand had no impact of Cas12 activation, underscoring the role of target strand nicks in modulating Cas12a activation. Across the nicked regions of Type VI, full Cas12 activation was observed at positions P1–P8, P10, P15– P18, and P21–P24 ( Fig. 1D ). Notably, positions P9, P11, P13, P19, and P20 exhibited only partial activation, whereas nicking at positions P12 and P14 completely inhibited Cas12a trans cleavage activity ( supplementary Fig. S1F). To ensure that these observations are not a sequence-specific artifact of our model system, we expanded our study to include a novel spacer sequence, focusing on positions known to exhibit significant activation variability (P5, P8, P9, P10, P11, P16, P20, and P22; supplementary Fig. S1G, Table S3 ). As can be seen, the outcome was consistent with our initial observations across all six configurations (Types I to VI), supporting that the positional effects we see on Cas12 activation from single stranded breaks are generalizable and independent of the target sequence. To further understand the mechanism of Cas12a inhibition by single stranded breaks, we explored the local protein environments at the nicked positions using published Cas12a crystal structures (PDB: 8Y03) of Cas12 in complex with gRNA and dsDNA ( 33 – 35 ). With the hypothesis that single stranded breaks at certain positions may alter protein-DNA interactions and therefore affect Cas12a activation, we evaluated the structure at sites where we had seen clear inhibition of activity ( Fig. 1D ). Interestingly, we found the region most affected by single strand breaks (P9 to P14) aligns with the endonuclease domain (RuvC and BH), which is critical for both cis and trans cleavage. Table S4 summarizes the rest of the nick positions rendering substantially diminished activity of Cas12a, along with their interacting amino acids (within 3.5 angstroms). Similarly, we found that P19 and P20, which exhibit semi-muted activation upon nick introduction, primarily interact with amino acids in the REC2 domain, which is involved in gRNA binding to the protein ( 35 ). While the precise biochemical implications of these alterations remain to be fully explored, the collective data and structural observations provide new insight into the mechanisms regulating Cas12a activation and highlight the role of protospacer integrity. Importantly, these findings also present a novel perspective on how the CRISPR-Cas12 system can be manipulated at molecular level, introducing an opportunity to exploit these findings to advance the Cas12a diagnostic platform. Sensor development With these new design considerations in-hand, we set out to leverage our findings for the development of an updated Cas12a-based detection platform ( Method 4-5 ). To begin on sensor development, we assessed Cas12a activation across the 24 nicked positions (P1 to P24, Fig. 1D ) for configuration Types I through VI to identify an optimal design (nicking point) for sensor development. The Type VI system containing a single stranded break at P8 emerged as a promising candidate as this site produced complete activation of the Type VI system and negligible activation of Types I to V. This unique configuration enables an ‘AND logic’ sensing mechanism within our RAPID system, wherein full system assembly is required for Cas12a activation. Specifically, trans cleavage is triggered only when all nucleic acid components are present. Consequently, the Type VI configuration, provided the foundation for what will become the RAPID sensors. The RAPID Type VI system has the potential to provide a highly flexible diagnostics platform as enzyme activation can be triggered by multi-modal nucleic acid formats, including ssDNA, mRNA and miRNA, as well its conventional dsDNA target format ( Fig. 2A , light blue). CRISPR gRNAs (red) were engineered for sequence-specific binding of these diverse nucleic acid types, which, in the presence of a target sequence, leads to Cas12a trans cleavage of a fluorescence reporter. Download figure Open in new tab Fig. 2: System configurations of RAPID for nucleic acid detection. (A) Configurations showing PAM-free detection across various nucleic acid types: short and long single-stranded nucleic acids, and short (blunt-ended) and long double-stranded DNA (dsDNA). Dark blue represents the PAM duplex, red the gRNA, cyan the target strand, and orange the trans PAM. (B) Performance of RAPID in detecting various lengths of target ssDNA (28-nt, 89-nt, 139-nt) compared to PAM-containing dsDNA. The non-target controls (NTC) display higher a background signal with AsCas12a and low background with LbCas12a. (C) Detection capabilities of RAPID for target RNA lengths of 28-nt, 65-nt, and 116-nt, demonstrating better performance with shorter RNA. AsCas12a exhibits higher background noise with NTC compared to LbCas12a. (D) Use of RAPID for PAM-free detection of target dsDNA, including blunt-ended dsDNA (52-bp) and longer dsDNA (139-bp), compared to PAM-containing dsDNA. The NTC for AsCas12a shows high background noise, in contrast to LbCas12a, which shows low background noise. ( Method 5 ). All nucleic acid sequences are presented in Table S5 . n=3 technical replicates; bars represent the arithmetic mean ± SD. RFU is relative fluorescence unit. Another critical component of RAPID is the PAM duplex ( Fig. 2A , dark blue strands), a dsDNA sequence consisting of a PAM (orange) and neighbouring ssDNA toehold domain (dark blue, bottom right). The PAM sequences are introduced in trans configuration, meaning PAM sites are separate from the target nucleic acid, relieving the PAM sequence requirement from the diagnostics design process. Crucially, when short target ssDNA or miRNA bind to the toehold domain, a pseudo-nick is created at position P8 in the Type VI system, enabling Cas12a activation ( Fig. 2A (I)). Similarly, longer ssDNA or RNA target sequences can also trigger system activation and form a pseudo-nick at P8 ( Fig. 2A (II)). For the detection of PAM-free dsDNA, we have designed a universal PAM duplex that lacks a toehold domain ( Fig. 2A (III)). In this configuration, the PAM sequence is again introduced in trans and is structurally distinct from the dsDNA trigger. Here, we present two use cases for this dsDNA design. In the first, the gRNA-Cas12a complex binds to both the universal PAM duplex and a blunt-ended PAM-free dsDNA, leading to activation ( Fig. 2A (III)), and a second, where a segment of the gRNA binds to the universal PAM while another segment binds to an extended PAM-free dsDNA target, thereby activating Cas12a ( Fig. 2A (IV)). Importantly, the RAPID system directly detects PAM-free dsDNA, in contrast to a recent PAM-free dsDNA platform ( 30 ), where the dsDNA target must first undergo transcription to generate RNA amplicons for downstream detection ( 30 ). Nucleic acid sensing with RAPID We tested RAPID using two commercially available Cas12a orthologs, specifically Acidaminococcus sp. (AsCas12a) and Lachnospiraceae bacterium (LbCas12a). Our initial experiments focused on enhancing Cas12a detection sensitivity for ssDNA using RAPID by pairing ssDNA with a sticky end dsDNA containing the PAM duplex, as depicted in Fig. 2A (I and II), Method 5 . Notably, the dsDNA configuration enhances the trans cleavage signal of CRISPR-Cas12, likely due to its greater stability when complexed with the Cas12-gRNA, in contrast to the weaker signal observed with ssDNA ( 2 , 36 ). This property makes our Type VI system particularly interesting, as it mimics the dsDNA configuration while exhibiting an enhanced trans cleavage fluorescence signal. We experimented with short ssDNA target strands (light blue) of 28 nucleotides (nt), corresponding to Fig. 2A (I), and longer strands of 89 and 139 nt, corresponding to Fig. 2A (II). Utilizing PAM-containing dsDNA as a benchmark, we observed that the trans cleavage signals from all ssDNA triggers were comparable to those from PAM-containing dsDNA for LbCas12a and AsCas12a ( Fig. 2B ). Moreover, we observed that AsCas12a exhibited a higher no template control (NTC) background signal than that of LbCas12a, consistent with findings from previous studies ( 13 , 24 ). Extending this analysis to RNA targets, we discovered differential trans cleavage activation thresholds between the two Cas12a variants. RNA target sequences activated trans cleavage remarkably well when complexed with AsCas12a, while trans cleavage was significantly muted with LbCas12a ( Fig. 2C ). The target RNA lengths tested included a 28-nt sequence, akin to the configuration in Fig. 2A (I), and longer sequences of 65 and 116-nt, paralleling the setup in Fig. 2A (II). Notably, the shorter target RNA (28-nt miRNA) was more effective at triggering trans cleavage than longer RNA counterparts. Further exploration of the RAPID system revealed efficient detection of PAM-free dsDNA. We first tested a titration of the universal PAM duplex concentration in the detection of both blunt-ended and longer dsDNAs, finding the optimal concentration to be 200 nM ( supplementary Fig. S2A&S2B). Using this PAM duplex concentration, we next exposed the RAPID system ( Fig. 2A (III)) to 52 bp PAM-free target dsDNA sequence, finding PAM-free sequence could match or exceed activation of the PAM-containing dsDNA (∼10 bp) for both LbCas12a and AsCas12a ( Fig. 2D for 52 bp, supplementary Fig. S2C). We similarly assessed detection of PAM-free dsDNA at an arbitrary location in a 139 bp target, mirroring the configuration in Fig. 2A (IV), and again found activation of both Cas12a homologs on par or better than PAM-containing dsDNA ( Fig. 2D for 139 bp, supplementary Fig. S2C). Trans -cleavage activation with RAPID Conventionally, Cas12a systems are considered DNase systems designed to target DNA and enable DNA trans cleavage, as demonstrated by platforms like DETECTR and HOLMES ( 2 , 24 ). Therefore, Cas12a platforms have historically been developed for ssDNA substrates, with RNA-based trans cleavage exhibiting lower efficiency in traditional Cas12a applications ( 37 ). Here, with the aim of expanding Cas12a’s role in diagnostics, we investigate whether other genomic substrates can be harnessed for Cas12a trans cleavage ( Fig. 3A ). We began by designing a series of sequences to test trans cleavage efficiency with substrates comprising DNA, RNA, and chimeric (e.g. mixed sequences that contain both deoxyribo- and ribo-nucleotides) polymers (Reporters 1 to 16 in Table S6). Download figure Open in new tab Fig. 3: Optimization of reporter sequences for the RAPID system. (A) Schematic of the RAPID system configured to cleave DNA, RNA, and chimeric reporters modified with fluorophore-quenching pairs. (B) Performance of RAPID with AsCas12a. (C) Performance of RAPID with LbCas12a. The system includes 16 reporters, divided into four categories: DNA/RNA/chimeric polymers (R1 to R4), DNA homopolymers (R5 to R8), RNA homopolymers (R9 to R12), and chimeric homopolymers (R13 to R16). Reporters are annotated with repeat numbers indicating base repetition (e.g., (T) 6 = TTTTTT, (rUA) 3 = rUArUArUA). ( Method 6 ). All reporters are modified with 56-FAM and 31ABkFQ. Results are expressed as mean ± SD (n = 3) in relative fluorescence units (RFU). n=3 technical replicates; bars represent the arithmetic mean ± SD, all sequences can be found in Table S6. We first applied RAPID to trans cleave the traditional ssDNA sequence (R1: TTATT) using AsCas12a and LbCas12a ( Fig. 3B,C ). Consistent with conventional Cas12a systems ( 13 ), we noted a high non-specific signal in the absence of target sequence (fluorescence background) with AsCas12a compared to LbCas12a ( Fig. 3B (i),C(i)). We next applied RAPID to trans cleave an RNA sequence (R2: rUrUrArUrU, ( Fig. 3B (i)). The cleavage activity was stronger for AsCas12a than for LbCas12a, but the signal for both Cas12a homologs was largely muted. Subsequently, we tested modified RNA sequences by introducing DNA bases (R3: (rUA) 3 and R4: (ArU) 3 ). Surprisingly, the trans cleavage signal was stronger for these chimeric sequences than for the RNA-based sequences for both AsCas12a and LbCas12a ( Fig. 3B (i) and Fig. 3C (i)). This provided significant reduction of the background signal for AsCas12a with R3 ( Fig. 3B (i) for R3), in comparison to conventional DNA substrate R1 ( Fig. 3B (i) for R1). Importantly, in addition to minimizing background interference, this is the first instance where chimeric reporters have been shown to match the efficiency of DNA-based reporters in Cas12a-based systems. Building on these preliminary findings, we screened Cas12a RAPID DNase activity with a series of ssDNA homopolymer reporters in the presence and absence of target sequence [R5: (T) 6 , R6: (A) 6 , R7: (C) 6 , and R8: (G) 6 ]. As before, ssDNA homopolymers yielded very high background signals for R5, R6, and R7 with AsCas12a, compared to the high signal and low background observed for LbCas12a. Negligible trans cleavage was observed for poly G (R8) in either Cas12a ortholog ( Fig. 3B (ii) and Fig. 3C (ii)), which we anticipate is primarily due to photoinduced electron transfer from guanine, the most easily oxidized nucleobase, to the excited fluorophore ( 38 , 39 ). Next, we tested RAPID with an expanded series of RNA homopolymer reporters [R9: (rA) 6 , R10: (rU) 6 , R11: (rC) 6 , and R12: (rG) 6 )] to assess trans cleavage RNase activity. As expected for Cas12a, we saw a generally weak response, with no RNase activity observed for R9 and R12 in AsCas12a and for R12 in LbCas12a. Limited activity was seen for R10 and R11 with AsCas12a and for R9, R10, and R11 with LbCas12a ( Fig. 3B (iii) and Fig. 3C (iii)). Finally, we applied RAPID to trans cleave chimeric homopolymers [R13: (ArA) 3 , R14: (TrU) 3 , R15: (GrG) 3 , and R16: (CrC) 3 ]. The results showed good to excellent trans cleavage effects for R13, R14, and R16 with both Cas12a homologs, expanding the chimeric reporter toolbox, while R15 was not cleaved by either Cas12 enzyme ( Fig. 3B (iv) and Fig. 3C (iv)), perhaps due to the guanine’s photoinduced electron transfer ( 40 ). All sequences are listed in Table S6, and kinetic plots are available in supplementary Fig. S3A-C. The results showed that selected RNA-based reporters could be leveraged for diagnostics, albeit weakly. However, chimeric probes performed comparable to some ssDNA sequences, with enhanced performance using chimeric probe R3, which significantly decreased the non-specific background signal of AsCas12a ( Fig. 3B ), indicating that AsCas12a possesses a broader substrate tolerance for these variants than LbCas12a. Additionally, it was noted that the background signal from R1, which is the traditional reporter substrate for Cas12a, was significantly higher than R3 – the novel reporter for AsCas12a, aligning with observations from traditional CRISPR-Cas12a systems ( 24 , 41 ). Furthermore, ssDNA sequences demonstrated superior trans cleavage activity compared to their RNA counterparts when interacting with LbCas12a ( Fig. 3C (ii & iii)). This differential activity highlights the inherent preference of LbCas12a for ssDNA substrates within the RAPID system. In comparison to asCas12a, LbCas12a exhibited only slightly reduced ability for cleaving the chimeric probes, R3 and R4, and excellent cleavage of chimeric reporters R14 and R16, respectively, suggesting flexible trans cleavage substrate specificity within the RAPID platform. All the kinetic plots are presented in supplementary Fig. S3A-C. For LbCas12a, testing non-canonical reporter sequences also provided a productive strategy to improve the signal-to-noise ratio. Wanting to benchmark the performance of chimeric reporters, we then integrated CRISPR-Cas12a with an upstream recombinase polymerase amplification (RPA) reaction for a PAM-containing dsDNA target (synthetic HPV-18 – Table S7 ) and chimeric reporter (R3: (rUA) 3 ). We were pleased to find both LbCas12a and AsCas12a homologs, in combination with RPA, provided sensitivity down to 1 aM ( supplementary Fig. S4A&B, Method 6 ). Control reactions without RPA were run in parallel to identify the detection limit of the Cas12a homologs alone (10 pM target concentration). Ligation-induced DNA repair with RAPID Having introduced a strand break into the Cas12a DNA substrate, here we test whether in situ ligation of a DNA nick can restore trans cleavage activity ( supplementary Fig. S5A). To test this concept, we introduce a duplex probe phosphorylated at the 5′-end of the target strand to enhance ligation efficiency ( Fig. 4A ). We then selected four distinct nick positions—P5, P8, P9, and P12— corresponding to activated (P5, P8), semi-muted (P9), or completely muted (P12) RAPID configurations ( Fig. 1D ). For configurations where the strand break impairs trans cleavage (P9, P12), the addition of ligase to one-pot RAPID reactions completely restores activity ( Fig. 4B – green bars, supplementary Fig. S5B). As expected, both unimpaired RAPID configurations (P5, P8) do not benefit from the addition of ligase. The activity of these reactions was benchmarked against RAPID reactions with a nickless PAM-containing dsDNA substrate and a no-template control (NTC). Download figure Open in new tab Fig. 4: Ligation-Induced Trans Cleavage. (A) Schematic representation of nicked DNA repair using T4 DNA ligase. The duplex probe contains a 5’-end that is phosphorylated at the nicked point, enabling repair in the presence of ligase, which restores the trans cleavage signal in one-pot reaction format. (B) Results depicting ligation activity of nicked DNA at various positions. The green bar is post-ligation, while the blue bar is before ligation. Positions P5 and P8 demonstrate complete activation regardless of T4 ligase presence, corresponding to the primary discovery from P1 to P20 on the heat map in Fig. 1 . Position P9 exhibits partial activation without ligase, which is fully restored upon ligase addition. At position P12, no activation occurs without ligase; however, the signal is recovered with ligase. Nickless dsDNA with a PAM serves as a control template, and NTC represents the no-target control. ( Method 14 ). (C) Demonstration of miRNA ligation to a DNA substrate, forming a chimeric DNA-RNA configuration in a single reaction. As position P8 showed promising results for detecting RNA ( Fig. 2 driven by the addition of MgCl 2 ,), here, without additional MgCl 2 and with the presence of the miRNA target and T4 ligase, a strong signal is generated, whereas low signal is observed without T4 ligase, and no signal in the negative control lacking miRNA. ( Method 15 ). (D) Testing the limit of detection for miRNA-21 using ligation-induced trans cleavage, achieving significant detection at concentrations as low as 100 pM. (See inset; Method 15 ). Symbols (+) and (-) denote the presence and absence of ligase enzyme, respectively. NTC is no target control (negative control). Data represent mean ± standard deviation from n = 3 technical replicates at 60 min. All sequences are contained in Table S8. Beyond demonstrating a rescue of RAPID activity, we next considered the potential of ligase to enhance the detection of miRNAs with RAPID. By joining the target miRNA to a DNA substrate, a chimeric DNA–RNA substrate is formed to activate CRISPR-Cas12-mediated trans cleavage. We were excited to see that the addition of ligase enzyme (1.5 U) to RAPID detection of miRNA doubles the trans cleavage activity compared to no ligase addition ( Fig. 4C ). The resulting system, without pre-amplification, can detect miRNA-21 down to 100 pM with chimeric reporter 3 (R3) ( Fig. 4D inset , Method 15, Table S8). Compared to existing miRNA detection strategies, this ligation-based approach provides enhanced flexibility and superior sensitivity. miRNA detection with RAPID miRNAs are a category of small, single-stranded, non-protein-coding RNAs with a length of approximately 19-23 nucleotides, and have been widely reported as biomarkers of several cancers, diabetes, immune dysregulation, and other disease states ( 42 , 43 ). The direct detection of these biomarkers using conventional Cas12a systems is challenging due to incompatible reaction buffers with low salt concentrations, the requirement for a PAM, and high background signal ( 24 ). Leveraging this proof-of-concept, we next optimized the RAPID system components for the direct detection of miRNAs ( Fig. 5A ). This began with an evaluation of the MgCl 2 concentration, which we found was optimal at 31 mM. ( supplementary Fig. S6A). Next, titrations of duplex DNA, AsCas12a enzyme, and gRNA concentrations were screened, finding optimal performance at approximately 20 nM, 90 nM, and 90 nM, respectively ( supplementary Fig. S6B-D; heatmap - supplementary Fig. S6E ). We also found that the addition of ∼6% PEG-8000 as a crowding agent enhances the trans cleavage signal ( supplementary Fig. S7). Download figure Open in new tab Fig. 5: Utilizing RAPID for miRNA Diagnostics. (A) Schematic of the RNA detection setup using RAPID. (B) Limit of detection for miRNA-21 using RAPID, achieving sensitivity down to 97 pM ( supplementary Fig. S6F). (C) Heatmap illustrating orthogonality testing of RAPID for seven miRNAs: miRNA-21, miRNA-320, miRNA-210, miRNA-180, miRNA-193, miRNA-324, and miRNA-340. (D) Stability testing of freeze-dried RAPID components over 7 days, demonstrating good stability and field deployability. ( Method 8-9). All incubation time is 1 h. Sequences in Table S10. n=3 technical replicates; bars represent the arithmetic mean ± SD. NTC refers to no template control. With the optimized conditions in-place, we proceeded with the development of a RAPID assay for miRNA-21 ( Supplementary Fig. S8A), chosen for its significance in breast cancer prognosis ( 42 ). ( Method 8 ). Beginning with the use of RAPID alone, we found a detection limit of 97 pM ( Method 8 , Fig. 5B and supplementary Fig. S8B), which provides comparable performance to Cas13 RNA-based miRNA detection ( 44 , 45 ). This enhancement is particularly notable given that RAPID covers a 16 nt miRNA sequence, potentially translating to greater specificity, compared to the ≤12 nt coverage in earlier Cas12 systems with a detection limit within the nM range ( 23 , 24 ). Additionally, we tested RAPID’s specificity for seven miRNAs: miRNAs-320, 210, 187, 193, 324, and 340, which are biomarkers for other conditions, including cancer prognosis ( 42 ) — and confirmed orthogonality in detection ( Fig. 5C and supplementary Fig. S9A, Table S9). The isothermal nature of the RAPID platform positions it well as a tool for decentralized diagnostics, so we evaluated the performance of freeze-dried reagents for the potential distribution of test kits without a cold chain ( Method 9 ). As can be seen from endpoint reads (2 h), we found that the system demonstrated robust room temperature stability for up to one week from the time of freeze-drying ( Fig. 5D and supplementary Fig. S9B). Reactions containing all the RAPID components, including AsCas12a and R3 reporter, were flash frozen in liquid nitrogen and freeze-dried overnight in a Labconco FreeZone 6 Liter -84 °C Console Freeze Dryer and vacuum packed with desiccant for storage. Discrimination of single and two-point mutations with RAPID The capacity to identify point mutations in target sequences can provide an important advantage to diagnostics, enabling the detection of viral variants, the presence of drug resistance, or the detection of oncogenic mutations, among many other applications ( 46 ). Yet, detecting such subtle differences is currently a challenge ( 30 ). To investigate the potential of the RAPID platform to provide single nucleotide polymorphism (SNP) detection, we screened ssDNA sequences containing point mutations and paired two-base mismatches across the target binding regions (1-16, Fig. 6 ). The sequences, including the target regions (blue) and the mismatches (red), are detailed in Tables S11-12, respectively, facilitating a direct comparison between the mutated sequences and their wildtype (WT) counterparts. Download figure Open in new tab Fig. 6: Evaluating the Ability of RAPID to Detect Mismatches in DNA and RNA. (A) Detection of Single-Point Mutations in ssDNA: The left panel shows schematics of the point mutation, and the right panel displays the trans cleavage rate. (B) Detection of Two-Base Mismatches in ssDNA: Schematics of the mismatches are on the left, with corresponding detection results on the right. (C) Detection of Single-Point Mutation in miRNA-21: RAPID targets a 16-nt region within the protospacer of miRNA-21, with the remainder binding to duplex DNA. ( Method 7 ) Green bars represent the wild type; blue bars represent mutants and non-target controls (NTCs). Results are expressed as trans cleavage rate per min (for analysis, see Method 7 ). Bar numbering corresponds to mutation sites (e.g., G1T indicates guanine mutated to thymine at position 1, nearest to the trans PAM). n=3 technical replicates; bars represent the arithmetic mean ± SD. Final DNA concentration is 2 μM, while miRNA is 50 nM. Trans cleavage rate is analysed within 2 h. All data was collected as a time series. The trans cleavage rate data was analysed within 2 h (Method 7), while the 60-minute end point data is presented in Supplementary Fig. S10A. Tracking the rate of trans cleavage within 2 h, RAPID demonstrated a marked ability to distinguish point mutations located proximal to the trans PAM region for ssDNA (positions #1 to 10) termed “RAPID seed region” ( Fig. 6A , Supplementary Fig. 10A for endpoint measurement in RFU, Method 7 ). However, the detection efficiency waned for mutations situated more distal from the trans PAM (positions #11 to 16). Next, we introduced paired two-base DNA mismatches within the target sequences. As above, we saw strong selectivity in the RAPID seed region within a 60 min reaction time ( Fig. 6B , Supplementary Fig. S10B). The capacity for SNP detection in combination with PAM-free detection introduces an opportunity to rationally design detection schemes to place target mutations within the region of high selectivity for both SNP and paired mismatches. Importantly, these extended regions of specificity for SNPs and two-paired mismatches are not possible using the conventional CRISPR-Cas12a system containing ssDNA ( 2 , 30 ). The small size nature of many miRNAs, and their high sequence similarities, makes the discrimination of closely related targets especially challenging for CRISPR diagnostics ( 47 ). These small, single-stranded, non-protein-coding RNAs are proving to be biomarkers of several cancers, diabetes, immune dysregulation, and other disease states ( 42 , 43 ). Here, with the goal of demonstrating the potential of RAPID to address this need, we tested point mutations across synthetic miRNA-21 variants ( Fig 6C , Supplementary Fig. S10C) using AsCas12a and the R3 chimeric reporter. Interestingly, RAPID provided SNP selectivity in proximal ( 2 – 8 ) and distal ( 13 – 15 ) regions of the miRNA, highlighting differences in RAPID’s interactions with miRNA and ssDNA and suggesting Cas12a activation requirements are altered when accommodating non-canonical RNA substrates. RAPID’s sensitivity to nucleotide alterations (SNPs) parallels findings from SAHARA employing Cas12a systems for RNA detection ( 23 ). Importantly, RAPID covers 16-nt of the target sequence and is specific over a longer region compared to the recent studies from SAHARA that could only detect 12-nt of the target sequence with limited specificity ( 23 ). RAPID coupled with RT-LAMP for SARS-CoV-2 RNA detection in clinical samples To test the potential for RAPID to serve as an isothermal diagnostic alternative to gold standard reverse-transcription quantitative PCR (RT-qPCR), we developed a RAPID assay for the SARS-CoV-2 nucleocapsid gene. Here, demonstrating the flexibility of RAPID, we replaced RPA with reverse transcriptase loop-mediated isothermal amplification RT-LAMP; ( Fig. 7A , Method 10 ). To establish the protocol, we first tested RAPID separately with synthetic DNA strands that mimic the two loops of the anticipated LAMP dumbbell amplicon structure ( Fig. 7A and supplementary Fig. S11A), where RAPID converts the ssDNA into dsDNA via the duplex probe for enhanced trans cleavage activity. The results ( supplementary Fig. S11A) revealed that designing the system to target both ssDNA LAMP dumbbells could significantly enhance detection. With this result in hand, both loops were targeted, taking advantage for the first time, of the ssDNA switching to dsDNA with RAPID for enhanced trans cleavage performance. Download figure Open in new tab Fig. 7: RAPID Coupled with RT-LAMP for SARS-CoV-2 RNA Detection. (A) Diagram illustrating the interaction of RAPID with the loop region of the LAMP-generated dumbbell structure. (B) Proof of concept demonstrating detection in samples with 100 copies/µL (positive) and control (negative) samples, showcasing that RNA detection with RAPID and LAMP can be completed within 15 minutes. (C) Limit of detection data illustrating the ultra-sensitivity of RAPID with LAMP, detecting as low as 0.1 copies/µL within 30 minutes. (D) Bar graphs depicting the detection of different SARS-CoV-2 variants using RAPID coupled with LAMP, all within 30 minutes. (E) Data demonstrating RAPID’s selectivity against RNA from other viral targets. (F) Schematic illustrating the workflow from collection to detection of patient samples and detection using RAPID system coupled with LAMP. (G) Screening results for 25 patient samples with 14 negative and 11 positive, respectively. (H) Data presenting RNase P values for the 25 patient samples, distinguishing between positive and negative samples. (I) Plot showing fluorescence intensity of the freeze-dried RAPID-LAMP assay up to 7 days. Results after 2 hours of incubation at 37 °C ( Methods 11-12 ). n=3 technical replicates; bars represent the arithmetic mean ± SD. NTC refers to no template control. Using combined novel RT-LAMP and RAPID assay design, we tested full-length synthetic RNA representative of the SARS-CoV-2 (nucleocapsid gene) variants: Wuhan (Twist _102024), Alpha (Twist _103907), Beta (Twist _104043), Gamma (Twist_104044), Delta (Twist_104533), and Omicron (Twist_105204), finding detection of SARS-CoV-2 positive samples in about 5 minutes following a 30 min RT-LAMP pre-amplification reaction ( Fig. 7B and supplementary Fig. S11D, Tables S13-14). We next screened for the volume of RT-LAMP reaction, finding that 25 μL of the RT-LAMP reaction volume allowed for maximizing sample input ratio, while not inhibiting the RAPID reaction ( supplementary Fig. S11B). Accordingly, this volume was chosen for further experiments. Having established the conditions for viral RNA detection, we next evaluated the analytical sensitivity for SARS-CoV-2 (Wuhan) of the coupled RT-LAMP/RAPID reaction, finding detection of as low as 1 copy/μL ( Fig. 7C and supplementary Fig. S11C), comparable to standard RT-qPCR methods ( 48 , 49 ). Further testing confirmed RAPID’s ability to detect synthetic RNA corresponding to SARS-CoV-2 variants of concern (VOCs), including synthetic RNA encoding nucleocapsid alpha, beta, gamma, delta, and omicron, as well as the original Wuhan strain ( Fig. 7D , supplementary Fig. S11D, Table S14). Next, a selectivity study was performed to test RAPID’s capacity to discriminate synthetic SARS-CoV-2 target RNA sequences from other viral respiratory pathogens, including influenza virus (H1N1 and H7N9) and other coronaviruses (229E, NL63, OC43, MERS-CoV, and SARS-CoV-1) ( Fig. 7E and supplementary Fig. S11E). We next set out to evaluate the diagnostic performance of RT-LAMP/RAPID using clinical samples from suspected cases of SARS-CoV-2 infection collected in Toronto, Canada. ( Fig. 7F , Methods 11-12 ). A total of 25 nasopharyngeal samples were analysed using the RAPID diagnostic platform in parallel with RT–qPCR (U.S. CDC protocol ( 50 )) with RAPID, in general, providing comparable accuracy to the gold standard method ( Fig. 7G &H, Tables S16). Of the 25 patient samples tested for SARS-CoV-2, four samples with Ct values >33 demonstrated false negative, reducing accuracy to 84.00% (95% confidence interval [CI], 63.92% to 95.46%) (Table S17). However, for samples with Ct values ≤33, the RT-LAMP/RAPID was 100% (95% CI, 83.89% to 100.00%) accurate compared to RT-qPCR (Table S16-18), supporting a role for RAPID in providing isothermal and low-burden screening of viral RNA targets. To ensure the RNA integrity of the patient samples and as a criterion for sample inclusion in this phase of the project, patient RNaseP measurement was performed using RT-qPCR ( Fig. 7H ), with Ct values for RNaseP ranging from 28.8 to 37.2 (Table S16). Extending our early work with lyophilization of RAPID for miRNA detection, here, individual freeze-dried RAPID and RT-LAMP reactions were stored at room temperature to evaluate shelf stability ( Fig. 7I and supplementary Fig. S11F&G). We tested the system stability up to one week, with the 2-hour endpoint reported (37 °C). The results were comparable to previously reported CRISPR-based freeze-drying ( 51 , 52 ). While these results validate the potential of RAPID for field use, they also suggest that further improvements could enhance its long-term stability beyond 1 week. We anticipate that by using commercial lyophilization processes to produce freeze-dried pellets and provide more effective packaging methods, the combined system has the potential for deployment as a decentralized medical diagnostic. Altogether, RAPID represents a significant leap in CRISPR-based diagnostics. DISCUSSION In summary, we have discovered that the presence of a nick on the target strand of dsDNA can modulate Cas12a activity, with the effect—activation or deactivation—depending on the nick’s location ( Fig. 1 ). It is possible that this phenomenon mirrors the natural immune defense process, wherein the gRNA-guided Cas12a system not only identifies the protospacer sequence but also verifies its integrity. By introducing breaks or nicks, disruptions in the sequence of the target strand can lead to trans cleavage-controlled activation when bound to Cas12a ribonucleoproteins. We have also demonstrated, for the first time, that in situ ligation of pre-existing nicks can fully restore Cas enzyme activation signals ( Fig. 4 ). Building upon this insight, we have developed a PAMmer system that enables PAM-free targeting of nucleic acids by modifying both the spacer length and altering its nucleic acid structure ( Fig. 2 ). With the PAMmer system, we achieved for the first time a universal approach to target dsDNA compatible with both Cas12a orthologs: AsCas12a and LbCas12a, without traditional PAM constraints. We found that dsDNA could be recognized at blunt ends in a PAM-independent manner ( Fig. 2A (III), 2D). Moreover, our system also proved capable of detecting PAM-free dsDNA within a sequence ( Fig. 2A (IV), 2D), adding an important new tool for directly targeting PAM-free dsDNA. Our results indicate that AsCas12a exhibits greater versatility than LbCas12a in targeting various nucleic acid types, facilitated by the PAMmer system. Thus, AsCas12a can effectively target both RNA and DNA, while LbCas12a shows a preference for DNA ( Fig. 2B-D ). This contrasts with previous work ( 26 , 27 ), which demonstrated PAM-free detection via toehold activation and temperature modulation with Cas12a. While Cas12a traditionally shows weaker RNase activity compared to DNase activity, we enhanced the signal-to-noise ratio for AsCas12a by engineering RNA sequences to incorporate interspaced DNA base modifications, creating chimeric sequence reporters. This modification significantly improved the signal intensity and reduced background noise, which varies between AsCas12a and LbCas12a, with the former generally exhibiting higher background levels ( Fig. 3 ). The use of chimeric probes has thus proven, for the first time, to be an enabling method for boosting the analytical sensitivity of CRISPR-based diagnostics. Capitalizing on these advancements, we introduced RAPID—a sensitive and selective diagnostic method that can detect ssDNA, dsDNA, miRNA, and long RNA without PAM restrictions. Enhanced by chimeric sequence reporters, RAPID achieves picomolar level sensitivity for direct detection of miRNAs ( Fig. 6 ), rivaling the RNA-targeting capabilities of the Cas13 system ( 44 , 45 ). Importantly, conventional Cas12a and Cas13 systems do not directly detect point mutations in ssDNA and RNA, respectively ( 28 , 30 ). RAPID, however, exhibits improved and robust selectivity for single- and double-point mutations in ssDNA and can discriminate point mutations in miRNA at several sites, making it suitable for SNP genotyping ( Fig. 5 ). Combined with isothermal pre-amplification via RT-LAMP, RAPID reaches PCR-level performance within 40 minutes (10 min post 30 min RT-LAMP reaction) ( Fig. 7B ). Clinical validation of RAPID using SARS-CoV-2 patient samples showed complete concordance with RT-qPCR results for samples with Ct values ≤33 ( Fig. 7G ). Finally, the RAPID system, leveraging RNA-guided nucleic acid targeting, effectively trans cleaves chimeric reporters, broadening our understanding of the mechanisms by which the Cas12a system recognizes and acts upon genomic materials in trans cleavage contexts. By overcoming traditional PAM constraints, RAPID significantly expands the potential of CRISPR-based tools in diagnostics. We look forward to seeing how these new strategies for Cas12a are adopted by others to expand the use of CRISPR-Cas systems in research and in the promotion of distributed health care. Materials and Methods Reagents and materials All oligonucleotides including the duplex probes used in this work were synthesized by Integrated DNA Technologies (IDT). All modified oligos were purified by HPLC, while unmodified oligos were only subjected to standard desalting. Acidaminococcus sp (AsCas12a) was purchased from IDT (#10001272). Lachnospiraceae bacterium, LbCas12a (#M0653T), Cas12 diluent (#B0653A), Cas12 reaction buffer (NEBuffer™ r2.1, #B6002S), and RNase inhibitor (#M0314L) were purchased from New England Biolabs (NEB). Magnesium chloride solution (#7786303) was obtained from Sigma Aldrich. Polyethylene glycol/dimethyl sulfoxide solution 50% (w/v) was purchased from Sigma Aldrich (#P7306). DNase/RNase free deionized water (#10977015) from Thermo Fisher Scientific was used in all experiments. Assembly of nicked DNA activators (Method 1) To investigate the effect of nicks on the target strand of dsDNA (target strand: top strand of dsDNA that binds to the gRNA), we assembled three DNA oligonucleotides, designated as NTS, TS Fragment A and TS Fragment B. NTS is the non-target strand while TS is the target strand. DNA oligos were assembled in a 1X phosphate-buffered saline solution and heated to 95 °C for 5 min before cooling down to room temperature to facilitate formation of nicked dsDNA. Nicked DNA activators with nicks at 24 different target points were systematically designed by keeping the NTS constant while varying TS Fragments A and B. All sequences are presented in Table S1. Additionally, nicks on the NTS were also tested following the same annealing process. Sequences in Table S2. We also tested another nick containing dsDNA to establish generality of concept (see Table S3 for sequences). All sequences are contained in Table S1. CRISPR-Cas12a assay (Method 2) Following the annealing process, CRISPR-Cas12 assay components were prepared in final concentration of 1X NEB 2.1 buffer to a final reaction volume of 40 µL, adjusted with nuclease-free water. Final concentrations of assay components were as follows: 50 nM LbCas12a, 50 nM gRNA, 40 U RNase inhibitor, 125 nM DNA reporter and 10 nM annealed dsDNA. This mixture was aliquoted in 10 µL volumes into a 96-well plate to enable four technical replicates per condition. Reactions were monitored for 2 hours at 37 °C using the Roche LightCycler 480 II. Fluorescence data at 100 min were normalized and analyzed to assess nicking effects ( Fig. 1 and Fig. S1). Sequences of all nucleic acids used are listed in Tables S1 to S2. To confirm the generality of our findings, additional experiments employing a different gRNA (gRNA_02) within some regions exhibiting tunable Cas12 activation were conducted using the same protocol. Results from these experiments are presented in Figure S1G, and the corresponding nucleic acid sequences are provided in Table S3. Preparation of nucleic acid targets including RNAs (Method 3) All dsDNA were synthesize using gBlock of ssDNA obtained from IDT. T7-containing ssDNA templates for RNA including miRNA and crRNA transcription along with the primers were also purchased from IDT. 40 nM of the T7-containing ssDNA template was amplified using Q5 PCR kit (NEB #M0491) to form the T7-containing dsDNA. RNAs were synthesized using the HiScribe T7 High Yield RNA Synthesis Kit (NEB # E2050) by spiking in the amplified DNA templates into the reaction, and the reaction was performed at 37 °C from 4 to 16 h. The resulting reaction was treated with 4 units of DNase I (NEB #E2050 - M0303AVIAL) for 15 mins at 37 °C before purification with the Monarch RNA Cleanup Kit (NEB #T2040) and quantification with the NanoDrop One spectrophotometer (Thermo Fisher Scientific). Gel electrophoresis (Method 4) Cas12a trans cleavage of ϕX174 virion DNA in the presence of nicked dsDNA fragments was evaluated by gel electrophoresis. dsDNA fragments with nicks along the target strand, TS_02 - where 02 represent another dsDNA sequence, at positions P8, P12, P13, and P21 were prepared by the annealing process detailed previously. CRISPR-Cas12 assays included 90 nM LbCas12a, 90 nM gRNA, 20 U RNase inhibitor, 31.25 mM MgCl 2 , ϕX174 virion DNA (NEB N3023S), and 10 nM of annealed nicked dsDNA all in 20 µL 1X NEB r2.1 buffer. The reaction was then incubated for 30 minutes at 37 °C. Reaction mixtures (20 µL) were resolved by 0.8% agarose gels stained with SYBR™ Safe (Thermo Fisher Scientific S33102). Electrophoretic separation was conducted at 120 V for 1 hour and imaging was performed using a Bio-Rad ChemiDoc imaging system. The results of these experiments are presented in supplementary Fig. S1D. All nucleic acid sequences used in these experiments can be found in Table S1. Similar experiments were conducted using dsDNA activators with unperturbed target strands (TS_02) and non-target strands nicked at positions S9, S11 and S16 (NTS_02). Results from these experiments are shown in Fig. S1E. All sequences for these experiments are listed in Table S2. RAPID assay (Method 5) ssDNA and RNA Detection. A 50 µL reaction mixture was prepared containing the following components: 1X r2.1 NEB buffer, 90 nM LbCas12a (or AsCas12a), 50 U RNase inhibitor, 20 nM duplex probe, 90 nM gRNA, 31.25 mM MgCl₂, 500 nM ssDNA reporter (R1), and 50 nM ssDNA or RNA trigger. From this mixture, 15 µL was aliquoted into a 384-well clear reaction plate (Fisher Scientific 4483285) for technical triplicates. The plate was centrifuged for 2 minutes at 2000 × g and 4 °C using. Following centrifugation, the plate was transferred to a QuantStudio 5 real-time PCR system (ThermoFisher Scientific), where fluorescence was measured over a period of 2 hours at 37 °C. dsDNA Detection. A 50 µL reaction mixture was prepared as follows: 1X r2.1 NEB buffer, 90 nM LbCas12a (or AsCas12a), 50 U RNase inhibitor, 200 nM universal PAM, 90 nM gRNA, 31.25 mM MgCl₂, 500 nM ssDNA reporter (R1), 5.81% PEG8000, and 50 nM PAM-free dsDNA trigger. Similar to the ssDNA/RNA detection setup, 15 µL of this reaction mixture was aliquoted into a 384-well clear reaction plate. The remaining steps, including centrifugation and fluorescence measurement, were carried out as described above. All nucleic acid sequences are detailed in Table S5. Fluorescence reporter screening and RPA experiments (Method 6) Reporter screening : to screen the 16 different reporters (including DNA/RNA/chimeric sequences, DNA homopolymers, RNA homopolymers, and chimeric homopolymers) used in this study, a 50 µL reaction mixture was prepared as follows: 1X r2.1 NEB buffer, 90 nM LbCas12a (or AsCas12a), 50 U RNase inhibitor, 20 nM duplex probe, 90 nM gRNA, 31.25 mM MgCl₂, 10 nM 89-nt ssDNA trigger, and 125 nM of the various reporter types . From this mixture, 15 µL was aliquoted into a 384-well clear reaction plate, following the same procedure as described for ssDNA and RNA detection. The remaining steps, including centrifugation and fluorescence measurement, were performed in the same manner. All sequences are contained in Table S6. Cas12a RPA experiments : the RPA-Cas12a reaction is performed in two steps: ( i ) RPA amplification. The RPA pre-amplification was set up starting with TwistAmp® Basic at 37 °C for 20 min. 280mM MgOAc was added to trigger reaction before incubation. Each reaction was 20 μL containing 14 mM MgOAc final concentration. ( ii ) 2 μL of RPA product was added to 18 μL of Cas12a reaction (LbCas12a and AsCas12a) for both conventional ssDNA and chimeric reporters. The CRISPR Cas12a master mix consisted of 1X NEB 2.1 buffer, 50 nM Cas12a, 1U/μL RNase inhibitor murine, 50 nM crRNA and 125 nM ssDNA or chimeric reporters. The fluorescence was then measured every 30 s using Roche Lightcycler 480 II at 37 °C for 30 min. miRNA-21 detection (Method 8) Cas12a reactions were assembled by mixing AsCas12a and gRNA at final concentrations of 90 nM in 1X NEB r2.1 buffer supplemented with 31.25 mM MgCl 2 , 5.81% PEG 8000, and 1U/µL murine RNase inhibitor (NEB). The fluorophore-quencher reporter (R3) and duplex probe (dsDNA co-activator) were added to the Cas12 reaction at final concentrations of 500 nM and 20 nM, respectively, along with the various concentrations (at 20 µL) of the miRNA target in a 50 µL reaction volume on ice. Reactions were transferred into optical 384-well clear plates (Fisher Scientific #4483285) and incubated at 37 °C for 2 hours while the FAM fluorescent intensity was measured every minute using the QuantStudio 5 RT-PCR System. All oligos are listed in Table S10. Limit of detection of miRNA-21 calculated from 3σ/S, where σ is the standard deviation of the background signal and S is the slope of the fluorescence of target concentration line. Lyophilization (Method 9) Master mixes for all reaction components including Cas12 proteins, guide RNAs, reporter probes, buffers and salts were prepared as described above, excluding the sample (either miRNA or RT-LAMP amplicons as applicable) and PEG for miRNA reactions. 25 mg/mL each of sucrose and dextran were added to all freeze-dried reactions as a lyoprotectant as described previously ( 51 ). Reactions were flash frozen in liquid nitrogen and freeze-dried overnight in a Labconco FreeZone 6 Liter -84 °C Console Freeze Dryer (catalog #710611200) and vacuum packed with desiccant for storage. Mismatch detection in ssDNA and miRNA (Method 7) All templates containing single or double mismatches in ssDNA were obtained from IDT. A 50 µL reaction mixture was prepared containing the following components: 1X r2.1 NEB buffer, 90 nM LbCas12a, 50 U RNase inhibitor, 20 nM duplex probe, 90 nM gRNA, 31.25 mM MgCl₂, 500 nM ssDNA reporter (R1), and 10 nM or 2 µM ssDNA with mismatches or WT. From this mixture, 15 µL was aliquoted into a 384-well clear reaction plate (Fisher Scientific #4483285) for technical triplicates. The plate was centrifuged for 2 minutes at 2000 × g and 4 °C. Following centrifugation, the plate was transferred to a QuantStudio 5 real-time PCR system (ThermoFisher Scientific), where fluorescence was measured over a period of 2 hours at 37 °C. We also challenged RAPID’s selectivity using relatively higher and lower oligonucleotide concentrations and found similar performance for both point mutations and paired two-base mismatches ( supplementary Fig. S10B&C). RNA mismatches were tested the same way, except 5.81% PEG 8000 and chimeric reporter (R3 in place of R1) were added into the reaction and AsCas12a was used instead of LbCas12a. (Sequences in Tables S11-12). Trans cleavage rate calculation : Background subtracted fluorescent kinetic curves were fit to an exponential association model, following the equation y=A*(1-exp(-k*x)). The rate constant k, representing the trans cleavage rate, is plotted for each condition in triplicate. RT-LAMP assay (Method 10) RT-LAMP reactions using the WarmStart LAMP 2X Master Mix (E1700S, NEB) were assembled in 30 μL containing 0.2 μM for F3 and B3 primers, 1.6 μM for FIP and BIP primers, 0.4 μM for LF and LB primers, and 1.0 μL of template (water, RNA extracted, or in vitro transcribed RNA) was used per reaction. These primers targeted the nucleocapsid protein ( Supplementary Data Table S13). All reactions were prepared on ice, incubated at 61 °C for 30 minutes, then inactivated at 80 °C for 5 minutes. RT-LAMP/RAPID reaction for SARS-CoV-2 detection (Method 11) After the RT-LAMP reaction, 25 µL of the reaction mixture was added to the RAPID reaction mix to achieve a final volume of 50 µL. The final concentrations of the components in the RAPID-LAMP reaction were as follows: 1X r2.1 NEB buffer, 90 nM LbCas12a, 50 U RNase inhibitor, 20 nM PAM duplex 1, 20 nM PAM duplex 2, 90 nM gRNA1, 90 nM gRNA2, 31.25 mM MgCl₂, 500 nM ssDNA reporter (R1), and 25 µL of the RT-LAMP assay, making up the final volume of 50 µL. From this mixture, 15 µL was aliquoted into a 384-well clear reaction plate (Fisher Scientific #4483285) in technical triplicates. The plate was centrifuged for 2 minutes at 2000 × g and 4 °C. Following centrifugation, the plate was transferred to a QuantStudio 5 real-time PCR system (ThermoFisher Scientific), where fluorescence was measured over a period of 2 hours at 37 °C. Viral RNA extraction and RT-qPCR for SARS-CoV-2 detection (Method 12) Nasopharyngeal swab specimens were tested for positivity and analyzed by RT-qPCR, according to a protocol established by the U.S. Centers for Disease Control and Prevention (CDC) (Ct value ≤ 40 was determined to indicate a positive sample) ( 50 ). Viral RNA was isolated from patient samples using QIAamp Viral RNA Mini Kit (Qiagen, 52906) and RT-qPCR reactions were conducted using the QuantiNova Probe RT-PCR Kit (Qiagen, 208354) according to the manufacturer’s instructions. Briefly, 10 μL reactions were prepared in a 384-well plate format. Primers and probes for SARS-CoV-2 N and RNase P genes are listed in Supplementary Table S15-12 and were synthesized by IDT. All reactions were performed using the Applied Biosystems QuantStudio 5 Real-Time PCR Systems (Applied Biosystems, USA). Patient sample collection and ethical statement (Method 13) Nasopharyngeal swabs were obtained from 25 suspected individuals with respiratory disease from the clinical diagnostics lab of Mount Sinai Hospital (MSH), Toronto, Canada. This study was approved by the University of Toronto research ethics board (under human ethics protocol number 39531, which permitted the screening of SARS-CoV-2 virus samples. All experiments were conducted in accordance with relevant regulations and guidelines. Nicked DNA Repair (Method 14) DNA fragments corresponding to various nicked positions were prepared by annealing an equimolar mixture of three complementary fragments. The annealing was conducted at 95°C for 4 minutes in an annealing buffer [20 mM Tris-HCl (pH 7.5), 150 mM KCl, 1 mM EDTA, and 50 mM MgCl2], followed by a gradient cooling step to 4°C at a rate of 0.1°C/s. A 50 µL reaction mixture was prepared containing 1× T4 DNA ligase buffer, 1.5 U of T4 DNA ligase enzyme, 90 nM LbCas12a, 90 nM Lb gRNA, 50 U RNase inhibitor, DNA reporter 3, and 50 nM of the annealed DNA fragments. From this mixture, a 15 µL aliquot was dispensed into a clear-bottom black microplate for triplicate fluorescence readings. Trans cleavage activity was monitored at 37°C for 60 minutes using a BioTek plate reader, as previously described. The T4 DNA ligase enzyme was procured from Promega (Catalog #: M1801). miRNA-DNA Ligation (Method 15) For miRNA-DNA ligation, two DNA fragments and the miRNA target were annealed under the same conditions described above for nicked DNA repair. The reaction components and conditions were identical to the nicked DNA repair protocol. For both reactions, the T4 DNA ligase enzyme was excluded in conditions where ligation was not required. This method allowed for precise monitoring of trans cleavage activity under ligation and non-ligation conditions, enabling the evaluation of nick repair and miRNA detection. For limit of detection assay, 20 μL of various miRNA-21concenration is added to the reaction mix, while every other thing remains the same. Data Availability The data that support the plots within this paper and other findings of this study are available from the corresponding authors upon reasonable request. Approval for Public Release This work was cleared by DARPA and approved for public release, distribution unlimited. Further questions should be channeled to DARPA via their Public Release Center located at 675 N. Randolph Street, Room 03-028, Arlington, VA 22203-1714. Author contributions I.A.I. and F.L. contributed equally and conceived the ideas. K.P., I.A.I., F.L., A.C., J.D., J.N., and G.L. designed, performed, and analysed the experiments. X.L. contributed in shaping initial ideas. I.A.I. guided all experiments. I.A.I, F.L., S.S., J.D., A.C., S.F.J.D.S., and G.L. wrote the manuscript. S.F.J.D.S. handled all clinical trials. T.M. provided all the patient samples. A.A.G. provided useful insights to miRNA sensing. Y.Z. handled Cas structural analysis. I.A.I., F.L., A.C., S.J.D.S., J.D., S.S., Q.M., J.R.J.V., R.Z., P.B., M.S., K.B., M.C., S.P., X.L., J.P.T., Y.Z., K.P., and A.A.G. discussed the results, revised or commented on the manuscript. S.Y. and Z.L. supervised the phase I of the work. K.P. edited the manuscript and supervised the rest of the work. Ethics declarations Competing interests The authors declare the following competing interests: I.A.I., F.L., K.P., A.C., S.Y., and L.Z. are listed as inventors on a patent application related to the CRISPR-Cas12 PAM-free nucleic acid detection through target sequence breaks, involving the University of Toronto - Canada and the Hong Kong University of Science and Technology (US Patent App. 63/737,199). Additionally, F.L., I.A.I., A.C., S.Y., and K.P. are listed as inventors on a provisional patent related to enhanced RNA trans cleavage of Cas12 family for programmable nucleic acid detection, involving Orange Biotech Co., Hong Kong, and the University of Toronto, Canada with US Patent App. 63/633,180. The two patents are directly related to the reported work. K.P. and A.A.G. are co-founders of En Carta Diagnostics, Inc. The remaining authors declare no competing interests. Data availability The data that support the plots within this paper and other findings of this study are available from the corresponding authors upon reasonable request. Supplementary information Supplementary Figs. 1-7 and Supplementary Tables 1-15 Acknowledgement I.A.I. was supported by the Precision Medicine Initiative (PRiME) at the University of Toronto with internal fellowship number PRMUHT2024-001, and Canadian Institutes of Health Research (CIHR) with fellowship number 202410MFE-531769-419793. J.R.J.V. was supported by the Ontario Graduate Scholarship (OGS). This work was generously supported by funds to K.P. from the NSERC Discovery Grants Program (RGPIN-2016-06352); the Canadian Institutes of Health (CIHR) Foundation Grant Program (201610FDN-375469); and CIHR Project Grant (CIHR 202403PJT-520192-BE2-CEAA-129834; and support through the Canada Research Chairs Program (Files 950-231075 and 950-233107), and Early Researcher Award, Round 15, from the Ontario Ministry of Colleges and Universities. We would also like to acknowledge the support to A.G. and K.P. through the Smart Noninvasive Assays of Physiology Program (4500004932(518829)) from the Defense Advanced Research Projects Agency (DARPA), Contract No. N66001-23-2-4042. The views, opinions and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the Department of Defense or the U.S. Government. The authors appreciate Prof. Alexander Green at Boston University, USA for his initial support in miRNA detection and helpful discussion. Special thanks to Profs. Zhigang Li and Shuhuai YAO at the Hong Kong University of Science and Technology for their supervision and discussions at the initial stage of the project. I.I. thanks Abasi-ifreke Karena Idorenyin, Zachary Iwe, and Ariel Iwe for their understanding and support during this work. Footnotes ↵ * Co-first Authors References 1. ↵ N. Mohammad , L. Talton , Z. Hetzler , M. Gongireddy , Q. Wei , Unidirectional trans-cleaving behavior of CRISPR-Cas12a unlocks for an ultrasensitive assay using hybrid DNA reporters containing a 3′ toehold . Nucleic Acids Res . 51 , 9894 – 9904 ( 2023 ). OpenUrl CrossRef PubMed 2. ↵ J. S. Chen , E. Ma , L. B. Harrington , M. Da Costa , X. Tian , J. M. Palefsky , J. A. Doudna , CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity . Science . 360 , 436 – 439 ( 2018 ). 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