key: cord-1014038-nd7ub2u3
authors: Wang, Meng; Zhang, Rui; Li, Jinming
title: CRISPR/cas systems redefine nucleic acid detection: Principles and methods
date: 2020-07-08
journal: Biosens Bioelectron
DOI: 10.1016/j.bios.2020.112430
sha: c34d3123caac8810712cd383d09970c26b7b00f2
doc_id: 1014038
cord_uid: nd7ub2u3

Methods that enable rapid, sensitive and specific analyses of nucleic acid sequences have positive effects on precise disease diagnostics and effective clinical treatments by providing direct insight into clinically relevant genetic information. Thus far, many CRISPR/Cas systems have been repurposed for diagnostic functions and are revolutionizing the accessibility of robust diagnostic tools due to their high flexibility, sensitivity and specificity. As RNA-guided targeted recognition effectors, Cas9 variants have been utilized for a variety of diagnostic applications, including biosensing assays, imaging assays and target enrichment for next-generation sequencing (NGS), thereby enabling the development of flexible and cost-effective tests. In addition, the ensuing discovery of Cas proteins (Cas12 and Cas13) with collateral cleavage activities has facilitated the development of numerous diagnostic tools for rapid and portable detection, and these tools have great potential for point-of-care settings. However, representative reviews proposed on this topic are mainly confined to classical biosensing applications; thus, a comprehensive and systematic description of this fast-developing field is required. In this review, based on the detection principle, we provide a detailed classification and comprehensive discussion of recent works that harness these CRISPR-based diagnostic tools from a new perspective. Furthermore, current challenges and future perspectives of CRISPR-based diagnostics are outlined.

1 Effective identification of the presence of specific nucleic acid targets, as well as 2 their sequence alterations, is vital for the accurate diagnosis and appropriate 3 management of cancer, infections and genetic diseases. For example, ALK (anaplastic 4 lymphoma kinase) gene rearrangement testing has been recommended for patients 5 with nonsquamous non-small-cell lung cancer (NSCLC) and is a prerequisite for 6 targeted therapy with crizotinib(Ettinger et al. 2018). The most effective method for 7 early identification of coronavirus disease (COVID-19) has been established by 8 examining the pathogenic sequences in respiratory tract samples, and such methods 9 are critical for reducing the spread of disease or managing individual 10 patients(Adhikari et al. 2020). Implementation of prenatal screening and diagnosis by 11 analyzing genomic abnormalities in circulating fetal DNA and amniotic fluid cells is 12 the surest way to reduce birth defects(Gray and Wilkins-Haug 2018; Malan et al. 13 2018). Therefore, nucleic acid detection that incorporates molecular-level information 14 is critical for the effective management of disease. Among numerous factors that can 15 facilitate the detection of target nucleic acids, proper detection methods are of prime 16 importance because a high-performance diagnostic test is a necessary prerequisite for 17 achieving reliable and timely results. Generally, the ideal nucleic acid detection 18 method should be sensitive, specific, rapid, portable, cost-effective and easy to use. 19 proteins to recognize and cleave targets complementary to guide sequences (namely, 1 spacers) of crRNAs (Fig. 1) . Distinct from restriction enzymes and traditional gene 2 editing tools, such as transcription activator-like effector nucleases (TALENs) and 3 zinc-finger nucleases (ZFNs), the RNA-guided cleavage of the Cas effector enables 4 more flexible utilization of this tool through a simple redesign of spacer sequences for 5 different targets and does not require the modification of the Cas effector. 6 Depending on the architecture of effector integration, CRISPR systems can be 7 divided into two classes: class 1 systems function via multi-effector cascades, and 8 class 2 systems rely on single-effector proteins (Makarova et al. 2015) . Possessing 9 remarkable simplicity and operability, CRISPR effectors from class 2 systems are 10 particularly sought after for a variety of purposes, and CRISPR/Cas9 is highly 11 popular (Shmakov et al. 2017 ). The Cas9 effector guided by its single-guide RNA 12 (sgRNA) specifically recognizes DNA targets that harbor a cognate 20-bp protospacer 13 with a downstream protospacer-adjacent motif (PAM) to induce R-loop formation and 16 Notably, the introduction of D10A or H840A silencing mutations into the RuvC or 17 HNH domain results in a programmable Cas9 nickase (Cas9n) that can generate a 18 nick at the corresponding strand of the target, whereas simultaneous inactivation of 19 both domains will lead to a nuclease-deficient Cas9 (dCas9) protein while 20 maintaining targeted reorganization capacity ( including facile biosensing assays (Li et al. 2019b ), rapid in situ labeling and efficient target enrichment for sequencing analysis, based on their RNA-guided targeted 1 recognition and cleavage activities. 2 Two other single-component class 2 Cas effectors, Cas12 and Cas13, display 3 RNA-guided targeted recognition and cleavage of double-stranded DNA (dsDNA) 4 and RNA, respectively. However, upon selectively targeting cognate sequences, 5 Cas12 and Cas13 effectors undergo a conformational change and display collateral 6 cleavage of nearby single-stranded DNA (ssDNA) and RNA, respectively(Chen et al. (Fig. 1) . This non-canonical collateral cleavage 8 activity of Cas12 and Cas13 effectors radically deviates from the canonical target 9 recognition and cleavage activity of the Cas9 proteins that turn to an inactive state 10 after targeted cleavage. Due to these unexpected activities, both Cas12 and Cas13 11 effectors can function as elegant signal amplifiers in detecting nucleic acids by 12 translating the presence of specific sequences into multiturnover nuclease activities, 13 thereby resulting in strongly elevated analytical sensitivity. Moreover, this 14 transduction process can be completed rapidly under mild conditions, which is highly 15 desirable for user-friendly utilization in both clinical and field settings. 16 Collectively, these new applications of CRISPR systems potentially represent 17 ideal candidates for high-performance diagnostic tools. In this review, we discuss 18 recent representative works exploiting CRISPR-based diagnostic tools for human The RNA-guided target recognition activities, which are derived mainly from 7 Cas9 variants, have enabled orthogonal tools of high innovation for pathogen and 8 disease detection, thus providing vital complements to methodologies currently used 9 in clinical practice. In this study, we classify these tools into three categories based on 10 types of Cas effectors: Cas9, dCas9 and Cas9n. A comparison of different Cas9 1 variant-based strategies for detecting nucleic acids is listed in Table 1 . 3 Well-confirmed theories suggest that CRISPR/Cas9 specifically scans its targets 4 in a strict PAM-dependent manner and then creates a double-strand break at the third 5 base upstream of PAM (Knight et al. 2015) . Thus, CRISPR/Cas9 can be used to 6 distinguish single base mismatches located in the PAM region. For example, Pardee 7 and colleagues proposed a Cas9-based Zika variant genotyping method with 8 single-base resolution, termed NASBACC, in which the Zika RNA sequences were 9 preamplified by nucleic acid sequence-based amplification (NASBA)(Pardee et al. 10 2016). The resulting amplicons were applied to trigger the toehold switch of specially 11 designed RNA hairpins and untie the LacZ ribosome binding site (RBS) and start 12 codon motif (AUG) that were otherwise folded and inactivated, thereby initiating 13 enzyme LacZ translation and colorimetric readout. In the NASBA step ( Fig. 2A) , 14 RNA amplification was accomplished via three procedures operating in series, during 15 which dsDNA products were intermediates. In the presence of the PAM sequence and 16 an adjacent target site, dsDNA intermediates could be cleaved by the Cas9/sgRNA 17 complex, resulting in a blocked NASBA reaction and thus no endpoint single output.

short fragments that functioned as primers to initiate the EXPAR process, resulting in 1 abundant ds-and ssDNA amplicons (Fig. 2B) . The robust exponential amplification 2 capability of EXPAR imparted outstanding analytical sensitivity to CAS-EXPAR to 3 0.82 aM. In terms of specificity, CAS-EXPAR can discriminate a single-base 4 mismatch located exactly at the cleavage site because the mismatch would hinder 5 primer extension of EXPAR, thereby eliminating readout. In addition, CAS-EXPAR 6 has successfully been applied to Listeria monocytogenes mRNA detection. However, 7 regarding miRNA detection, challenges remained due to their small size at ~19−23 (Fig. 2C ). By simultaneously adding orthogonal sets of padlock probes, 17 sgRNAs and TaqMan probes cognate with miRNAs of interest into a single reaction, 18 RACE has achieved multiple detection of up to three homologous miRNAs (miR-21, 19 miR-221, miR-222). 20 Compared with the abovementioned biosensing platforms, which require tedious 21 enzymatic steps or sophisticated equipment, the CASLFA method, which is a visual, 22 fast and field-deployed strategy, has been designed by integrating Cas9 detection into 23 a lateral flow format (Wang et al. 2020c) . In this scheme (Fig. 2D) , dsDNA targets 24 were first amplified using biotinylated primers by recombinase polymerase 25 amplification (RPA) or PCR, thereby generating biotinylated amplicons that could be 26 specifically recognized by Cas9/sgRNA to form ternary complexes. Upon trickling 27 onto the lateral flow device, these ternary complexes flowed laterally and hybridized 28 with the AuNP-DNA probe, an Au affinity-labeled oligonucleotide capable of binding 29 to the loop region of sgRNA, which resulted in the formation of quadruple complexes that were captured by the precoated streptavidin at the test line, while excess 1 AuNP-DNA probes spread forward and finally accumulated at the control line via the 2 immobilized capture probes, collectively providing a chromogenic indication for the 3 presence or absence of DNA analyte. Performing EGFR gene detection as a proof of 4 concept, CASLFA has exhibited the ability to enable rapid (entire test completed 5 within 1 h) detection over a wide temperature range of 20~37 with minimal 6 equipment. Although this method is easy to use, the performance is not reduced 7 because CASLFA detected microbe genomic DNA with a sensitivity reaching 8 150~200 copies/reaction and distinguished L. monocytogenes from orthogonal 9 foodborne pathogens with few cross-reactions. Therefore, this technology offers an 10 attractive option for rapid nucleic acid detection in a simple format, which is 11 particularly relevant for point-of-care testing (POCT) and nonlaboratory 12 environments. flow assay that enables fast and field-deployed detection. 5 2.1.2 dCas9-mediated biosensing assays 6 The dCas9 system, a programable DNA-binding tool that can be repurposed only 7 by changing the spacer sequence of sgRNA, functions as an anchor that delivers 8 various effectors, such as fluorescence and enzymes, to desired loci or as a catcher 9 that captures target DNA for further manipulation and transduction, thus providing 10 orthogonal tools for nucleic acid detection. One of the typical examples is a 11 dCas9/sgRNA-SG I-based strategy proposed by Guk's group for methicillin-resistant 12 Staphylococcus aureus (MRSA) detection (Guk et al. 2017) . In this strategy, the 13 dCas9/sgRNA complex was repurposed to bind the mecA gene, a MRSA-specific gene responsible for methicillin resistance, and then pulled down the genomic DNA 1 that was subsequently visualized using SYBR green I (SG) staining (Fig. 3A ). This 2 strategy eliminates nucleic acid separation steps by directly using cell lysates, 3 indicating its superiority to traditional methods in which DNA extraction and 4 purification are required before PCR amplification (Hagen et al. 2005) . Verified by 5 testing a series of clinical isolates, this strategy enabled rapid detection within 30 min 6 with a limit of detection (LOD) down to 10 CFU/ml. In this system, an RNA-guided luciferase was constructed by fusing a pair of dCas9 16 proteins with the N-and C-terminal half of the firefly luciferase enzyme, and 17 dimerization was required for wild-type luciferase activity. Therefore, the fusion 18 dimers were reconstituted and catalyzed luminescence for fluorescent readout only 19 when there were two proximate protospacers spaced at a certain length apart along the 20 target sequence (Fig. 3B ). This PC reporter system has high specificity because its 21 output is premised on strict sequence and spatial restrictions. Another embodiment of 22 this principle of dimerization is in the development of the RCA-CRISPR-split-HRP 23 (RCH) method, a novel miRNA detection platform that used, in this case, the 24 split-horseradish peroxidase (HRP) system (Qiu et al. 2018) . In this platform, miRNAs 25 acted as primers to initiate RCA using the dumbbell-shaped DNA ring as templates, 26 which enabled discrimination between miRNA orthologs with similar sequences due 27 to the high specificity of the toehold-exchange reaction underlying the hybridization 28 of miRNA primers with dumbbell-shaped templates (Fig. 3C) . Compared with the 29 traditional RCA, amplicons of toehold-initiated RCA consisted of hundreds of tandem DNA hairpins, and the double-strand stem regions could recruit dCas9 protein pairs 1 that carried split-HRP, resulting in the generation of reconstituted HRP activity that 2 could initiate the TMB color reaction as a highly specific second-stage amplification. 3 Using let-7a as a model of detection, this platform exhibited a single-base specificity 4 and femtomolar sensitivity attributed to the double enhancement from both the 5 toehold-initiated RCA and proximate reconstitution of the split-HRP system. In addition to enzyme-based schemes, dCas9-mediated detections also involve 13 multidisciplinary fundamentals, such as electrochemistry, hydrodynamics, and 14 microfluidics, which has facilitated the development of amplification-free nucleic acid 15 tests. For example, combined with the highly sensitive graphene-based field-effect 16 transistor (gFET) sensor, a dCas9-based handheld device, termed CRISPR-Chip, has 17 been fabricated for simple, rapid and selective on-chip electrical assays of The reorganization of DNA targets by dCas9/sgRNA complexes significantly reduced 16 their electrophoretic mobility to a level lower than the defined critical mobility due to 17 the relatively large molecule size of dCas9/sgRNA/DNA ternary complexes, resulting 18 in migration and accumulation toward the nanojunction, whereas untargeted DNA 19 species moved in the opposite direction, toward the bulk reservoir (Fig. 4C ). In this 20 format, the concentration and separation of dCas9/sgRNA-captured target DNA from 21 free DNA were simultaneously achieved, thereby enabling amplification-free 22 detection of target DNA in a direct and optical manner. 8 RNA-guided Cas9 nickase, which was previously exploited for precise gene Moreover, as a programmable nickase, the dissociation of the Cas9n effector from its 3 target will leave a nick at the desired site, which is the ready substrate for strand 4 extension and displacement reaction. These intriguing and delicate mechanisms form 5 the basis of Cas9n-mediated nucleic acid detection. 6 As a typical Cas9n detection scheme, the CRISPR-Cas9-triggered nicking PAM-out orientation, thereby generating a pair of exposed single-strand DNA with 12 free 3′-hydroxyl termini. Subsequently, a specially designed initiating primer (IP) pair 13 carrying overhanging 5′ Nb.BbvCI nickase recognition motifs were applied to 14 hybridization with exposed single-strand DNA at each border of the target. Catalyzed Although the above CRISDA scheme suggests an innovatively designed strategy 8 for isothermal amplification and ultrasensitive detection, the involvement of complex single-molecule sensitivity due to the efficient exponential amplification mechanism 6 and single-nucleotide specificity because of the intrinsic properties of Cas9 effectors. formamide's carcinogenicity and reproductive toxicity and the high cost of the probes. 18 Thus, additional efforts should be made to develop superior approaches. 19 CRISPR-Cas9 currently holds great promise for rapid, cost-effective and facile FISH (Table 2 ). However, due to the promiscuous 27 off-target binding and nonspecific adherence from the diversity of applied 28 dCas9/sgRNA complexes, CASFISH have failed to efficiently visualize nonrepetitive elements of strong clinical significance, thereby leading to limited application. 1 Clearly, an efficient signal amplification system should be introduced instead of 2 dCas9 tiling for nonrepetitive sequence visualization. To this end, a mechanism that 3 guarantees analytical specificity must be coupled to prevent the background signal Table 2 ). The formed DNA ring, in turn, acted as a template to initiate the 13 RCA reaction, resulting in localized signals of fluorescent spots from target elements 14 (Fig. 6D) . It is worth noting that CasPLA has single-base resolution because it 15 requires two indispensable Cas9-mediated target-binding events that are of low 16 tolerance to mismatches in the seed region of sgRNA. In theory, any genetic elements 17 carrying a pair of PAMs in proximity can be visualized and counted with such a 18 scheme, but only approximately 60% of potentially accessible genomic DNA targets 19 are detected (Fig. 6E) , which is probably limited by inefficient DNA ring circulation Table 2 ). Notably, three adjacently targeted sgRNAs are sufficient for 10 efficient imaging of individual transcripts due to the specificity of the CRISPR-dCas9 11 system and signal amplification capacity of the MS2 system, thereby relatively 12 simplifying the scheme design of this method. functioned as not only a programmable scissor but also an anchor to densely tile the 12 target region followed by pull-down using streptavidin magnet beads (Fig. 7B ). The 13 highly intensive sgRNA tiling with increments of approximately 20 bp contributed to 14 uniform enrichment, which endowed CRISPR-Cap with the ability to estimate allele 15 frequency and gene copy number. Notably, the elimination of hybridization capture 16 and PCR amplification (attributed to the large amount of DNA input during library 17 preparation) remarkably shortened the reaction time over parallel methods (Table 3) . 18 Metagenomic sequencing might suffer from a compromised sensitivity due to the 19 pronouncedly lower-level nucleic acids of infectious agents than that of hosts by Table   2 3), which could be translated into higher usability of sequencing space and sensitivity, 3 as well as lower sequencing depth required for nonhuman sequences. These 4 characteristics are critical for the early identification of infectious diseases in a 5 cost-effective manner. 6 Later, the same group published a novel targeted enrichment system termed The utility of RNA-guided target-recognition-triggered collateral cleavage as an 8 amplifier for the presence of nucleic acid targets has enabled numerous ultrasensitive 9 and portable approaches that potentially hold significant value for rapid and 10 field-deployed tests. We classify these approaches into two categories according to the 11 types of Cas proteins used: Cas13-based approaches and Cas12-based approaches. A 12 comparison of Cas13-and Cas12-based strategies for nucleic acid detection is listed 13 in Table 4 . (Table 4 ). In addition, SHERLOCK has been used for effective genotyping of Cas13-based microfluidic electrochemical device for miRNA detection. 5

In light of the notable boom in Cas13-based diagnostics, the establishment of a 6 complete workflow from sample to answer, which could be paramount to practicable 7 deployment, particularly in field settings, is highly valuable. To provide a feasible be ready for direct mixture with Cas13 detection reagents, thereby bridging raw 12 samples and the SHERLOCK assay without nucleic acid purification steps. Various bodily fluidsincluding urine, whole blood, plasma, serum, and saliva-were 1 compatible with this procedure of heat and TCEP/EDTA treatment within 10-25 min 2 while maintaining the test capacity of SHERLOCK, thereby minimizing instrument 3 requirements for Cas13-based diagnostic workflow (Fig. 8C) . To date, these three 4 consecutively published Cas13-based platforms (SHERLOCK, SHERLOCKv2 and 5 HUDSON) indicate the prospects of developing a simple, sensitive, specific, 6 cost-effective and portable diagnosis method for wide use that extends from epidemic 7 monitoring to tumor diagnosis and from clinical routine to field deployment. 8 Notably, although SHERLOCK has shown excellence in the detection of 9 pathogens, human genomic DNA and cfDNA, no attempt has been made to detect However, the real-time PCR system was integral for the generation and calculation of 16 the initial reaction rate, and this step was less flexible and might add to the complexity 17 of this method. To manufacture a portable and easy-to-use platform, another 18 amplification-free miRNA detection scheme using a microfluidic electrochemical 19 device that consists of an immobilization area and an electrochemical cell is activity at a catalytic rate of ~1250 turnovers per second upon binding to 12 guide-complementary dsDNA activators, which underlies the use of Cas12a as a 13 biosensor and amplifier for nucleic acid assays (Fig. 9A, Fig. 9B ). Among these however, it did not elucidate whether DETECTR could be employed for SNP and 28 RNA testing, which were highly desirable for broad diagnostic applicability.

amplification as a substitute for RPA and published almost at the same time as 1 DETECTR, has been proposed with the ability to detect SNP, even if the site resides 2 at the PAM-distal end . Such a high specificity is provided by the utility 3 of truncated crRNAs (16-nt and 17-nt guide sequences) that can enhance the cleavage 4 specificity of the CRISPR-Cas12a system (Lei et al. 2017 ). Using specially designed 5 primers to introduce PAM sequences during PCR preamplification, HOLMES can, in 6 theory, detect any sequence in a PAM-independent manner. In addition, HOLMES 7 enables RNA detection via the addition of reverse transcription steps that convert 8 RNA targets into cDNA before preamplification and detection. Notably, although 9 HOLMES theoretically has fewer sequence restrictions and wider usage, it is still not 10 an isothermal test and requires multiple steps. An enhanced version, namely, 11 HOLMESv2, will be discussed in the third paragraph of this part. has been proposed for the extended application of Cas12a diagnostics. In this method 2 (Fig. 10A) , modified ssDNA, designed with a methylene blue (MB) tag at the 3′ 3 terminus as the redox label and a thiol group at the 5′ terminus for covalent 4 immobilization onto the gold electrode, was employed as the electrochemical reporter. 5 In the presence of targets, collateral cleavage activity of Cas12a was activated to 6 cleave the ssDNA reporters, thereby unleashing the MB tag from the gold electrode 7 surface and resulting in a peak current. Therefore, the electrical signal could be 8 monitored as a specific proxy for the presence of DNA targets. In this format, structure not only brought the MB tag closer to the electrode, thereby increasing the 16 electron transfer rate constant, but also fully exposed the single-strand loop region of 17 hairpin DNA to interference with Cas12a cleavage, collectively leading to an 18 improved detection sensitivity (Fig. 10B ). In addition to electrochemistry technology, 19 microfluidics and nanotechnology have also been integrated with the Cas12a platform, 20 and a magnet-assisted V-chip (MAV-chip) has been fabricated for multiple detection 21 and quantification of SNVs (Shao et al. 2019) . In this work (Fig. 10C) , 22 catalase/platinum nanoparticles (PtNPs), conjugated with magnetic beads by the 23 ssDNA linker, was introduced as a signal transducer, which could be degraded by 24 DNA-target-triggered collateral cleavage activity of Cas12a to release PtNPs. By 25 applying the reaction system onto the volumetric bar-chart chip and using a magnet to 26 pull down magnetic beads, PtNPs were separated and then slid into the hydrogen 27 peroxide reaction wells to generate oxygen that could push red ink forward. In the 28 absence of the target, PtNPs were pulled down together with magnetic beads, 29 therefore, oxygen would not be generated and ink advancement would not occur. Therefore, the distance of ink movement could be measured to quantify of the DNA 1 targets. Employing a multichannel setting and well-designed crRNA, this method 2 enabled the multiple detection of cancer-related SNPs with allelic fractions as low as 3 0.01% without amplification. The discovery of more orthogonal Cas12 effectors exhibiting collateral cleavage RT-LAMP to directly amplify and subsequently detect target RNA isothermally at 15 55°C, and it omits the intervening reverse transcription step and thereby provides an 16 elegant solution for simple RNA detection with the Cas12b system (Fig. 9C) . In 17 addition, HOLMESv2 has been proven to have the ability to accurately quantify DNA 18 targets, as well as their methylation modification, collectively providing opportunities 19 to technologically expand their scope of detection to diverse utilities. However, this 20 method also has some disadvantages, such as high reaction temperature, sequence sensitive dsDNA detection with single-base specificity was developed (Fig. 9D) .

Although the observation that AaCas12b (Teng et al. 2018 ) performs 1 DNA-recognition-triggered collateral cleavage draws obvious parallels to that of 2 Cas12a orthologs, the high cleavage activity of the AaCas12b effector has endowed 3 CDetection with elevated signal amplification capability and improved sensitivity. 4 Concretely, CDetection presented a minimum LOD of 0.1 aM, which was revealed by 5 tests performed on HPV dsDNA diluted with human genomic DNA. Furthermore, 6 combined with tgRNA, a programmed gRNA that harbors a mismatch in the spacer 7 sequence that enables screening of targets with a single-nucleotide resolution, 8 CDetection could clearly discriminate between 3232A>G SNP sites and wild-type 2018), a multichannel all-in-one handheld device powered by tiny batteries to achieve 28 the whole process from sample to answer should be fabricated to enable real CRISPR single-base analysis at the single-cell level, but genomic DNA imaging, which is a 1 real concern, still suffers from inefficiency and failure to meet the standards required 

Practice Bulletin No. 162: Prenatal Diagnostic Testing for Genetic Disorders. 21

TALENs: a widely applicable technology for targeted genome 1 editing

Sources of PCR-induced distortions in high-throughput 3 sequencing data sets

In vivo genome editing with a small Cas9 6 orthologue derived from Campylobacter jejuni

High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects

Engineered CRISPR-Cas12a variants with increased activities and improved targeting 13 ranges for gene, epigenetic and base editing

Dynamics of 16 CRISPR-Cas9 genome interrogation in living cells

Increasing the specificity of CRISPR systems with engineered RNA secondary structures. characteristics of off-target sites bound by the Cas9 endonuclease

dCas9-mediated Nanoelectrokinetic Direct Detection of Target Gene for 4 Liquid Biopsy

CRISPR-Cap: multiplexed double-stranded DNA enrichment based on the CRISPR system. 7 Nucleic acids research

The CCTL

Cutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA 10 constructs in vitro

Fluorescence in situ hybridization: past, present and future

HOLMESv2: A 14 CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation 15

CRISPR-Cas12a-assisted nucleic acid detection

CRISPR/Cas Systems towards Next-Generation 19

Noise in gene expression determines cell fate in

An updated evolutionary classification of CRISPR-Cas systems

Effect of Cell-Free DNA Screening vs Direct Invasive Diagnosis on Miscarriage Rates in

Women With Pregnancies at High Risk of Trisomy 21: A Randomized Clinical Trial

CAS9 transcriptional activators for target specificity screening and paired 12 nickases for cooperative genome engineering

RNA-guided human genome engineering via Cas9

Target-enrichment strategies for next-generation sequencing

Comprehensive viral enrichment enables sensitive 3 respiratory virus genomic identification and analysis by next generation sequencing

A genome-wide 6 analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture

Rapid, Low-Cost Detection of Zika Virus Using Programmable 11

The next generation of CRISPR-Cas technologies 13 and applications

Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene 16 expression

Rapid and Fully Microfluidic Ebola Virus Detection with 19 CRISPR-Cas13a

Highly Effective and Low-Cost MicroRNA Detection with CRISPR-Cas9

FLASH: 5 a next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance 6 sequences

Imaging 8 individual mRNA molecules using multiple singly labeled probes

Double nicking by RNA-guided CRISPR 12 Cas9 for enhanced genome editing specificity

Evaluation of Hybridization 15

Capture Versus Amplicon-Based Methods for Whole-Exome Sequencing

High-Fidelity and Rapid Quantification of 18 miRNA Combining crRNA Programmability and CRISPR/Cas13a trans-Cleavage Activity

CRISPR-Cas12a Coupled with Platinum chemistry

Diversity and evolution of class 2 CRISPR-Cas systems

Rationally 6 engineered Cas9 nucleases with improved specificity

Direct observation 10 of individual endogenous protein complexes in situ by proximity ligation

Next-generation diagnostics: gene panel, exome, or whole 14 genome?

Clinical utility of 18 custom-designed NGS panel testing in pediatric tumors

CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base 1 specificity

Dimeric CRISPR RNA-guided FokI nucleases for highly 4 specific genome editing

Methods for Optimizing CRISPR-Cas9 Genome Editing 6

Genome editing 8 with engineered zinc finger nucleases

Ten years of next-generation 10 sequencing technology

Unconstrained genome 12 targeting with near-PAMless engineered CRISPR-Cas9 variants

RNAscope: a novel in situ RNA analysis platform for formalin-fixed, 15 paraffin-embedded tissues

CRISPR/dCas9-Mediated in Situ Imaging of mRNA Transcripts in Fixed Cells and Tissues

Rolling Circular Amplification (RCA)-Assisted CRISPR/Cas9

An RNA-Guided Cas9 Nickase

Method for Universal Isothermal DNA Amplification

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Mediated Lateral 6 Flow Nucleic Acid Assay

Receptor 2 Testing in Breast Cancer

American Pathologists Clinical Practice Guideline Focused Update

Genome-wide binding of 15 the CRISPR endonuclease Cas9 in mammalian cells

CRISPR/Cas12a-Mediated Interfacial Cleaving of Hairpin DNA Reporter for Electrochemical 18 Nucleic Acid Sensing

Dynamic DNA nanotechnology using strand-displacement 20 reactions

Single-Nucleotide Variation in mtDNA Using a CRISPR/Cas9-Mediated Proximity Ligation

Paired Design of dCas9 as a Systematic Platform for the Detection of Featured 5

Nucleic Acid Sequences in Pathogenic Strains

A CRISPR-Cas9-triggered 7 strand displacement amplification method for ultrasensitive DNA detection

• Discussion of the significance and basic principles of CRISPR-based diagnostics.• Cas9 variants have been applied to biosensing, imaging and sequencing.• Cas12 and Cas13 proteins have enabled ultrasensitive and portable diagnostic tools.• Discussion of challenges and perspectives of CRISPR-based diagnostics.

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: