An outbreak of betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 began in Wuhan, China in December 2019. COVID-19, the disease associated with SARS-CoV-2 infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR–Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated our method using contrived reference samples and clinical samples from patients in the United States, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT–PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement. SARS-CoV-2 in patient samples is detected in under an hour using a CRISPR-based lateral flow assay.

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CRISPR-Cas12-based detection of SARS-CoV-2.

Rapid detection of nucleic acids is integral to applications in clinical diagnostics and biotechnology. We have recently established a CRISPR-based diagnostic platform that combines nucleic acid pre-amplification with CRISPR-Cas enzymology for specific recognition of desired DNA or RNA sequences. This platform, termed specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), allows multiplexed, portable, and ultra-sensitive detection of RNA or DNA from clinically relevant samples. Here, we provide step-by-step instructions for setting up SHERLOCK assays with recombinase-mediated polymerase pre-amplification of DNA or RNA and subsequent Cas13- or Cas12-mediated detection via fluorescence and colorimetric readouts that provide results in <1 h with a setup time of less than 15 min. We also include guidelines for designing efficient CRISPR RNA (crRNA) and isothermal amplification primers, as well as discuss important considerations for multiplex and quantitative SHERLOCK detection assays.

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SHERLOCK: nucleic acid detection with CRISPR nucleases.

Dear Editor, Today, the need for time-effective and cost-effective nucleic acid detection methods is still growing in fields such as human genotyping and pathogen detection. Using synthetic biomolecular components, many methods have been developed for fast nucleic acid detection; however, they may not be able to satisfy specificity, sensitivity, speed, cost and simplicity at the same time. Recently, a very promising CRISPR-based diagnostic (CRISPR-Dx) (namely SHERLOCK) was established, which was based on the collateral effect of an RNA-guided and RNAtargeting CRISPR effector, Cas13a. SHERLOCK is of high sensitivity and specificity, and is very convenient in detection of target RNA. However, to detect DNA sequences, in vitro transcription of DNA to RNA must be conducted prior to the SHERLOCK test, which could be inconvenient. In a recent study, we found that Cas12a, which belongs to the class 2 type V-A CRISPR-Cas system, performed collateral cleavage on non-targeted ssDNAs upon the formation of the Cas12a/crRNA/target DNA ternary complex. Here, with the employment of this feature, we used a quenched fluorescent ssDNA reporter (e.g., HEX-N12-BHQ1 in Supplementary Table S1) as the probe, and developed HOLMES (an one-HOur Low-cost Multipurpose highly Efficient System), which could be used for fast detection of target DNA as well as target RNA. In HOLMES, if a target DNA exists in the reaction system, the Cas12a/crRNA binary complex forms a ternary complex with the target DNA, which will then trans-cleave non-targeted ssDNA reporter in the system, illuminating the HEX fluorescence (or any other fluorescence) (Fig. 1a). We ever purified ten Cas12a proteins (Supplementary Table S3) and found all showed the ssDNA trans-cleavage activity. To find the most suitable Cas12a for HOLMES (i.e., with high signal-to-noise ratios), we tested all ten Cas12a proteins and found Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a), Oribacterium sp. NK2B42 Cas12a (OsCas12a), Lachnospiraceae bacterium NC2008 Cas12a (Lb5Cas12a) and Francisella tularensis Cas12a (FnCas12a) showed good performance, among which LbCas12a was chosen for the following studies (Fig. 1b). To determine the sensitivity of HOLMES, we titrated target DNA, and found the minimum detectable concentration for Cas12a-crRNA was approximately 0.1 nM; however, when combined with PCR, the detectable concentration could be as low as 10 aM (Fig. 1c), which was comparable to the SHERLOCK system and was better than PCR alone or quantitative PCR using the SYBR Green method (Supplementary Figure S1). Therefore, to achieve higher sensitivity, PCR amplification was employed in the HOLMES test thereafter. To test whether HOLMES could discriminate singlebase differences, we made point mutations at different positions in the target DNA sequence, including both the PAM region and the guide sequences (Supplementary Figure S2a). When a full length of crRNA guide sequence (24-nt crRNA, Supplementary Table S2) was used, we found mutations in either the PAM sequences or the region of the 1st–7th bases of the guide sequence resulted in clear decline of the fluorescence signal; however, no significant difference was observed when the mutation was within the region of the 8th–18th bases (Supplementary Figure S2b), which was highly consistent with the previous report that the 5′-end seed region in the crRNA

/pdf/crispr-cas12a-assisted-nucleic-acid-detection-21kzn6w07q.pdf

CRISPR-Cas12a-assisted nucleic acid detection.

CRISPR-Cas systems provide microbes with adaptive immunity to infectious nucleic acids and are widely employed as genome editing tools. These tools use RNA-guided Cas proteins whose large size (950 to 1400 amino acids) has been considered essential to their specific DNA- or RNA-targeting activities. Here we present a set of CRISPR-Cas systems from uncultivated archaea that contain Cas14, a family of exceptionally compact RNA-guided nucleases (400 to 700 amino acids). Despite their small size, Cas14 proteins are capable of targeted single-stranded DNA (ssDNA) cleavage without restrictive sequence requirements. Moreover, target recognition by Cas14 triggers nonspecific cutting of ssDNA molecules, an activity that enables high-fidelity single-nucleotide polymorphism genotyping (Cas14-DETECTR). Metagenomic data show that multiple CRISPR-Cas14 systems evolved independently and suggest a potential evolutionary origin of single-effector CRISPR-based adaptive immunity.

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Programmed DNA destruction by miniature CRISPR-Cas14 enzymes.

CRISPR/Cas Systems towards Next-Generation Biosensing.

Dear Editor, The CRISPR-associated protein Cas12a (previously known as Cpf1), which is an endonuclease from the type V-A CRISPR system, has been applied in both in vivo genome editing and in vitro DNA assembly. Cas12a is guided by a single CRISPR RNA (crRNA) with a T-rich protospacer adjacent motif (PAM) sequence to cleave double-stranded DNA (dsDNA) targets, generating sticky ends. Different from Cas9, Cas12a cleaves both the target and non-target strands of a targeted dsDNA by a single active site in the RuvC catalytic pocket (Supplementary information, Figure S12a). Besides, Cas12a also processes precursor crRNAs to generate mature crRNAs. However, the cleavage activity of Cas12a on single-stranded DNA (ssDNA) targets is less understood. To investigate the ssDNA cleavage feature of Cas12a, we employed FnCas12a to cleave short ssDNAs that were labelled with 5(6)-carboxyfluorescein (FAM) on the 3′ terminus and found that the ssDNA cleavage sites were near the 22nd base (i.e., from the 21st to the 23rd), counting from the first 3′-base that was paired with the crRNA guide sequence (Supplementary information, Figure S1a and b and Tables S2 and 3). The cleavage did not require the existence of a PAM sequence in the targeted ssDNA (Supplementary information, Figure S1a and b). In addition, the same cleavage sites were obtained with crRNAs having guide sequences as short as 10 nucleotides (nt) (Supplementary information, Figure S1c and d), which indicates that Cas12a could cleave ssDNA at sites outside of the recognition sequence. We then tested Cas12a cleavage efficiency on ssDNA and dsDNA substrates, and cleavage of ssDNA was slower than that of dsDNA (Supplementary information, Figure S1e and f), whose PAM sequence may account for the higher efficiency. We also performed the Cas12a cleavage experiment with a ssDNA target labelled at its 5′ terminus (target-DNMT1-3-R-FAM5′). Surprisingly, no cleaved bands were observed at the predicted size (20 nt), but short (<6 nt) FAM-labelled products were generated (Fig. 1a). After careful analyses of experimental conditions, we found that only the ternary complex of Cas12a/ crRNA/targeted ssDNA (or targeted dsDNA) was able to cleave the 5′-labelled target ssDNA (target-DNMT1-3), generating short FAM-labelled products (Fig. 1b and Supplementary information, Figure S2). The ternary complex also promiscuously cleaved collateral ssDNAs that had no complementarity to the crRNA guide sequence in the reaction system, generating short products (Fig. 1c and Supplementary information, Figure S3). As it is difficult to distinguish the precise length of the short transcleavage products via polyacrylamide gel electrophoresis, the FAM-labelled short products were purified and analysed by liquid chromatography-mass spectrometry. The results showed that 5′-FAM-labelled substrates were mainly trans-cleaved to 4 nt, while 2-nt products were observed for 3′-FAM-labelled substrates (Supplementary information, Figure S4). We called the promiscuous cleavage of collateral ssDNAs transcleavage to distinguish it from the programmable on-target cleavage of target ssDNA (namely, cis-cleavage), and the proposed ssDNA cleavage processes were illustrated in Fig. 1d. When the ssDNA substrate was labelled at the 5′ terminus, the cis-cleaved 5′-labelled ssDNA products became collateral ssDNAs in the reaction system and were subsequently trans-cleaved into short products, explaining the observed cleavage pattern for 5′-labelled ssDNA substrate. We observed the trans-cleavage products in addition to the cis-products for short 3′-labelled targeted ssDNAs (Fig. 1a). The majority of the ternary complex most likely remained bound to the targeted ssDNAs after cis-cleavage, protecting the labelled 3′-terminus from exposing the trans-activity sites of the Cas12a ternary complex. Next, we tested nine randomly selected Cas12a proteins from different species in addition to the above tested FnCas12a (Supplementary information, Figures S5, 6a and Tables S1, 4 and 5), and all Cas12a proteins exhibited endonuclease activity on plasmid dsDNA (Supplementary information, Figure S6b), cis(Supplementary information, Figure S6c) and trans-cleavage activities on ssDNA (Supplementary information, Figure S6d). This indicates that the cisand trans-cleavage activities on ssDNA might be ubiquitous among Cas12a proteins. When shortened targeted ssDNAs were tested, complexes with 18-nt target ssDNAs that lacked a cleavage site also showed transcleavage activity (Supplementary information, Figure S7a), indicating that cis-cleavage was not a prerequisite for trans-cleavage activity. Trans-cleavage was implemented by the endonuclease activity of the complex, as circular ssDNA (M13mp18) could also be trans-cleaved (Supplementary information, Figure S7b). Moreover, we found that all tested Cas12a complexes except the AsCas12a complex had trans-cleavage activity on collateral dsDNAs (Figure S8), and the activity of the LbCas12a, BoCas12a and Lb4Cas12a complexes was much higher. To identify key residues involved in ssDNA cleavage of both targeted and collateral ssDNAs, we mutated several candidate residues in FnCas12a to alanines, including those related to the RNase activity (H843, K852 and K869) and those responsible for dsDNA cleavage (D917, E1006, D1255 and R1218) (refs. 1,7–9 and Supplementary information, Figure S9). Both cisand transcleavage of ssDNA were unaffected in the RNase activity-related mutants (Supplementary information, Figure S10), but the activities were completely lost or remarkably decreased with mutations in either the RuvC domain (D917A, E1006A and D1255A mutations in FnCas12a) or the Nuc domain (R1218A mutation in FnCas12a) (Fig. 1e, f). Recent studies showed that the RuvC catalytic pocket of both C2c1 and Cas12a was responsible for the cleavage of both strands of targeted dsDNA, leading us to propose that targeted ssDNAs were cis-cleaved by this catalytic pocket (Supplementary information, Figures S11a, b, d, e and S12c). Moreover, according to the structure of the C2c1-crRNAexcess DNA complex (Supplementary information, Figure S11c), trans-cleavage of collateral ssDNAs could also be achieved by the same catalytic pocket (Fig. 1g, and Supplementary information,

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CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA.

The clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 system has been widely applied in both transcriptional regulation and epigenetic studies. However, for multiple targets, independent expression of multiple single guide RNAs (sgRNAs) is needed, which is less convenient. To address the problem, we employed a DNase-dead Cpf1 mutant (ddCpf1) for multiplex gene regulation. We demonstrated that ddCpf1 alone could be employed for gene repression in Escherichia coli, and the repression was more effective with CRISPR RNAs (crRNAs) specifically targeting to the template strand of its target genes, which was different from that of dCas9. When targeting the promoter region, both strands showed effective repression by the ddCpf1/crRNA complex. The whole-transcriptome RNA-seq technique was further employed to demonstrate the high specificity of ddCpf1-mediated repression. Besides, we proved that the remaining RNase activity in ddCpf1 was capable of processing a precursor CRISPR array to simply generate multiple mature crRNAs in vivo, facilitating multiplex gene regulation. With the employment of this multiplex gene regulation strategy, we also showed how to quickly screen a library of candidate targets, that is, the two-component systems in E. coli. Therefore, based on our findings here, the CRISPR-ddCpf1 system may be further developed and widely applied in both biological research and clinical studies.

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Multiplex gene regulation by CRISPR-ddCpf1

In the original version of the Supplementary Information file associated with this Article, the site ‘rs5082’ was incorrectly given and should be corrected to ‘rs5028’.

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Qiu-Xiang Cheng

Papers

CRISPR-Cas12a-assisted nucleic acid detection.

CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA.

Multiplex gene regulation by CRISPR-ddCpf1

Erratum: Author Correction: CRISPR-Cas12a-assisted nucleic acid detection.