key: cord-0875152-muy75w79 authors: van Dongen, Jeanne E.; Berendsen, Johanna T.W.; Steenbergen, Renske D.M.; Wolthuis, Rob M.F.; Eijkel, Jan C.T.; Segerink, Loes I. title: Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities date: 2020-07-26 journal: Biosens Bioelectron DOI: 10.1016/j.bios.2020.112445 sha: 697f812db00ef7093bf50d80d88930b2f10a7f4f doc_id: 875152 cord_uid: muy75w79 With the trend of moving genomic test from (medical) laboratories to on-site testing, there is a need for nucleic acid based diagnostic tools combining the sensitivity, specificity and flexibility of established genomic diagnostics with the ease, cost effectiveness and speed of isothermal amplification methods. A promising new genomic sensing method is Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas)-based sensing. In this method Cas effector proteins are used as highly specific sequence recognition elements that can be combined with many different read-out methods for on-site point-of-care testing. This review covers the technical aspects of integrating CRISPR/Cas technology in miniaturized sensors for analysis on-site. We start with a short introduction to CRISPR/Cas systems and the different effector proteins and continue with reviewing the recent developments of integrating CRISPR sensing in miniaturized sensors for point-of-care applications. Finally, we discuss the challenges of point-of-care CRISPR sensing and describe future research perspectives. The access to rapid and reliable detection methods of nucleic acids is critical in many different fields, 55 such as life sciences, environmental monitoring, biotechnology and maybe most importantly health 56 care. Sensing of pathogens on the basis of their genetic information, for instance by monitoring 57 circulating cell-free DNA/RNA particles related to various types of diseases makes early diagnosis and 58 treatment possible. However, this depends on in methods that allow the detection of ultralow 59 concentrations of nucleic acids with high sensitivity and specificity. The currently ongoing COVID-19 60 outbreak, affecting millions of people around the globe, again shows the importance of early and 61 specific infectious disease detection for patient and health care in general, and for risk prevention of 62 further spread. 63 Established genomic diagnostic tools, such as polymer chain reaction (PCR), are superior in terms of 64 specific amplification of genomic sequences but come with several drawbacks in terms of usability 65 and costs (Scheler et al., 2014) . Most of the established nucleic acid sensing methods need complex 66 and bulky instrumentation, as well as trained personnel to operate them. The assays are costly to 67 perform due to multiple (manual) steps that are both time and material consuming. 68 Isothermal amplification methods have been developed (e.g. LAMP and RPA) that are faster than PCR 69 and can be operated at constant temperature, eliminating the need for sophisticated equipment like 70 thermocyclers (Martzy et al., 2019) . However, these new advantages come with tradeoffs in terms of 71 sensitivity and specificity. Even after optimization, single-nucleotide polymorphisms (SNPs) cannot 72 always be discriminated (Zanoli and Spoto, 2013) , while these small nucleotide changes will be 73 crucial in both pathogen and disease detection. 74 Therefore, there is a need for nucleic acid based diagnostic tools combining the sensitivity, specificity 75 and flexibility of established genomic diagnostics, with the ease, cost effectiveness and speed of 76 isothermal amplification methods. Such a newly developed diagnostic tool will be perfect for point-77 of-care (POC) testing. Point-of-care testing is defined as a (medical) diagnostic tool near or even at 78 the point-of-care, so bringing testing on site (Kost et al., 2008) . This on-site testing is accomplished 79 by integrating assays in (trans)portable devices. In ideal point-of-care testing, the steps one needs to 80 perform from raw sample to understandable result should be minimized, allowing unskilled 81 operators to perform analysis. This trend asks for total-analysis devices, integrating sample pre-82 treatment, target recognition and signal acquisition (sensing) in a single device. These total analysis 83 systems could comprise entire microfluidic chips, paper-based sensors or even single tube 84 reactions, as long as they are accompanied by easy protocols and accessible read-out methods. showing its versatility are appearing rapidly. In this paper we will introduce the CRISPR/Cas systems 113 and the different effector proteins, review the recent developments, discuss the challenges of POC 114 CRISPR sensing and describe future research perspectives. 115 2 Classification of CRISPR/Cas systems 116 Three decades ago, the first CRISPR/Cas systems were described in bacterial genomes (Ishino et al., 117 1987; Van Soolingen et al., 1993) . It took many years to show that CRISPR and Cas genes were part of 118 the adaptive immune system of bacteria, used as a defense mechanism against foreign nucleotides 119 (Bolotin et The crRNA sequence is customizable: by changing the spacer sequence present on the crRNA one can 157 change the genomic target sequence. However, sequence possibilities are limited by the effector 158 protein specific protospacer adjacent motif (PAM) sequence (yellow, figure 3 ). This PAM sequence 159 can be found upstream (from 5' -> 3') of the protospacer on the dsDNA. PAM sequence recognition 160 by the RNP complex originates from the task of CRISPR proteins in the adaptive immune system of 161 bacteria. The purpose of the CRISPR system is to protect bacteria from invading bacteriophages, 162 viruses. If the bacterium survives an infection, class 1 CRISPR effector proteins, together with Cas9 163 trim part of the viral DNA and add this to a so-called CRISPR array. In this way the bacterium creates 164 a library of previous infections to fight the viral infection in the future (Figure 2A ). This CRISPR array, 165 is transcribed and used for the formation of Cas9 RNP complexes, that screen for complementary 166 (viral) sequences ( Figure 2B ). However, the CRISPR array contains the same sequence as the viral 167 DNA. This is where the PAM sequence comes in: since Cas9 is involved in the protospacer 168 recruitment, this protospacer sequence is always followed by a Cas9 specific PAM sequence. Only if 169 this PAM sequence is present next to the target sequence, cleavage will take place. This is only the 170 case for the protospacer present on the viral DNA and not for the spacer on the CRISPR array (Mali et 171 al., 2013). 172 So, by the PAM sequence, which is a nucleotide combination of typically 2-4 bases which is not 173 present in the genome of the bacterium, bacteria can distinguish their own DNA from foreign DNA. 174 Besides this function, the PAM sequence also accelerates the search process for viral DNA in the 175 bacterium cell. Cas9 only has to unwind the DNA containing a PAM sequence . By unwinding the DNA, 176 complementation between spacer and protospacer can take place, initiating the enzymatic cleavage 177 properties of the effector protein, in this example Cas9. This cleavage results in blunt-end double 178 strand breaks in the target dsDNA ( Figure 3 ). Besides enzymatic cleavage, the Cas9 effector protein 179 possesses excellent sequence recognition. The enzymatically deactivated Cas9 effector protein 180 (dCas9) follows the same target recognition process but lacks the ability to perform blunt-end strand 181 breaks. 182 The PAM sequence is dependent on the type of Cas effector protein and the species it originates 183 from. PAM sequence recognition does not take place via the crRNA, but by the effector protein itself. 184 In the case of the commonly used spCas9 from Streptococcus pyogenes, the PAM sequences 185 following the protospacer is 5'-NGG-3' (where N could be any type of nucleotide DNA. Upon complementation between the RNP and a DNA sequence, the DNA sequence will be destroyed. To avoid cleavage of the CRISPR array, the PAM sequence is of importance. Only sequences followed by a specific PAM sequence will be recognized and will be cleaved by the RNP. any nucleotide base), can be found upstream (from 5'-> 3') of the protospacer sequence. This PAM 200 sequence is less crucial when sensing ssDNA, and it has been shown that enzymatic cleavage activity 201 can still be possible with mutated PAM sequences on the ssDNA target sequence . 202 While subtype Cas12a is more specific, with less off-target cutting compared to Cas9, the on-target 203 cleavage efficiency of Cas12a is also relatively low (Kim et 230 POC sensing is a rapidly fast-developing field in clinical diagnostics and is expected to define the 231 future of diagnostics in health care. POC sensors such as the handheld glucose sensors for diabetes 232 patients are widely known, but rapid development now also allows for sensors for the detection of 233 proteins, small (inorganic) molecules and nucleic acids. According to the World Health Organization 234 (WHO) POC testing needs to follow the ASSURED guidelines (Affordable, Sensitive, Specific, User-235 friendly, Robust and rapid, Equipment-free, Deliverable to all people who need the test) (Kosack et 236 al., 2017). According to these guidelines, it should be possible for a non-specialist to conduct and 237 interpret the test in a variety of settings, including low resource communities. CRISPR/Cas effector 238 proteins were previously used in nucleotide sensing applications and showed, in combination with 239 (s)gRNA, that they can specifically target nucleic acids with high efficiency and could therefore be 240 used in combination with POC devices for highly effective nucleic acid detectors. 241 As mentioned in the introduction, the first CRISPR sensing methods were designed using Cas13a 242 effector proteins (previously known as C2c2), which exerts two distinct RNAse activities. that still employ a nucleic acid amplification step in addition to the signal amplification obtained by 263 the effector proteins to further reduce the limit of detection. 264 In the next sections we will discuss CRISPR POC sensing methods based on their read-out mechanism: 265 Fluorescence, colorimetric or electronic. Table 2 gives an overview of all current POC CRISPR sensing 266 methods described in this review paper. 267 268 The first CRISPR sensing systems were based on an increase in fluorescence upon target sequence 269 recognition by the RNP complex. A major advantage of -fluorescence-based sensing is its 270 background-free sensing, drastically increasing the signal-to-noise ratio compared to other optical 271 techniques. The miniaturization of fluorometers enables performance of established fluorescence 272 based CRISPR sensing methods, like SHERLOCK, HOLMES and DETECTR on site. However, this 273 established fluorescence based CRISPR sensing techniques include numerous manual (pipetting), 274 and/or nucleotide amplification steps, besides there need of large volumes of (expensive) chemicals. 275 This compromises the user-friendliness and cost effectiveness of these established systems for POC 276 applications. In this section dedicated POC sensors based on CRISPR sensing will be reviewed that 277 reduce the number of manual steps, relatively easy and cheap to perform. and fast reaction times. The integrated fluorometer has a XYZ translation stage, which can be used to 296 move between detection reservoirs for rapid multiplexing. In their original design, 24 assays could be 297 run in parallel within 30 minutes. Due to the absence of an amplification step, no expensive 298 temperature regulation is needed and the price of 24 assays was calculated to be ∼$6 USD (Qin et 299 al., 2019). 300 He et al. developed an amplification-free POC system for rapid and accurate virus detection. The 301 African Swine Fever Virus (ASFV) is a DNA containing virus and can be sensed with CRISPR/Cas12a 302 effector proteins. Due to the high specificity and selectivity of the Cas12a-crRNA RNP complex, 303 differentiation between closely related viruses was possible, and a detection limit of 1 pM could be 304 reached within two hours for pre-treated DNAs. By increasing incubation time, improved detection 305 limits of 100 fM could be reached. For bodily fluids of infected animals, the LOD increases due to 306 autofluorescence. The chip formed a concentration boundary layer, creating an electric field gradient, and therefore a 326 spatial gradient in electrophoretic force which works against the drag force from the flow in the 327 channel. Since DNA, dCas9 and DNA-dCas9 complexes have different electrophoretic mobilities, the 328 authors could concentrate either the DNA-dCas9 complexes at a certain position in the chip (to pre-329 concentrate a specific DNA sequence before analysis) or both free DNA and DNA-dCas9 complexes 330 (for direct analysis by the use of fluorescent intercalating dyes (one vs two lines). The spacer region 331 on the crRNA can be designed to adjust the specificity and selectivity of the assay, down to 332 distinguishing SNPs. A downside of this system is that it can only be used for relatively short DNA 333 fragments (50-100 bp), as large DNA fragments (700-1000 bp) will not show enough mobility 334 difference on the addition of the relatively small dCas9. read-out of LFAs is simple and does not need sophisticated machinery, these assays require multiple 375 manual pipetting steps prior to the LFA step. Ideally, these steps could be minimized by incorporating 376 Cas sequence recognition and cleavage on the paper membrane, resulting a single dip of the assay in 377 the sample of interest prior to read-out. 378 based on so-called "toehold switches" to distinguish American and African Zika strains ( Figure 5A ). 417 Toehold switches are regulators that enable precise control over gene expression (Green et concentrations and assay steps we believe that this total reaction time can be reduced, and the 452 procedure could be simplified, enabling POC detection. 453 where the RNP complex acts as a target recognition element and the AuNPs are used for signal 464 amplification ( Figure 5C ). In the case of target recognition, Cas12a/13a initiates collateral cleavage of 465 all ssDNA/ssRNA, allowing degradation of the sequence needed for AuNP aggregation. Upon addition 466 of AuNPs, no aggregation will take place, indicating the presence of the target DNA. The absorbance 467 at 520 nm could be used to find a linear correlation between target sequence concentrations and red chamber. In the presence of target DNA, Cas12a will degrade the ssDNA tether, resulting in free 480 PtNPs in solution. By the sliding mechanism the free nanoparticles are transferred to the second 481 chamber, while a magnet retains the magnetic beads in the first chamber. If no target sequence is 482 present in the first chamber, the PtNPs are retained in the first chamber. In the second chamber, the 483 PtNPs catalyze the reaction of H 2 O 2 to O 2 , forming an overpressure that pushes detection-ink into the 484 readout channel, over a certain distance. The amount of PtNPs is related to the amount of O 2 485 produced and in this way the distance over which the ink travels can be related to the amount of 486 target sequence present in the first chamber. The platform can easily be adapted for multiplexing by 487 increasing the amount of parallel reaction chambers. Although only Cas12a was used as the sensing 488 enzyme, other Cas family proteins could also be applied in their platform by adjusting the tether 489 between the PtNP and the magnetic bead. The graphene-based field effect transistor (gFET) and the nanopore sensors do not use the cleavage 512 of an effector protein, but rather the binding of dCas9-crRNA complexes. 513 The gFET was the first sensor to employ electronic readout in CRISPR sensing. Using dCas9, Haijan et 514 al. use a gFET to obtain an electronic signal when a specific DNA sequence is present (Hajian et al., 515 2019). Their sensor is based on the principle that the proximity of a charged DNA molecule to the 516 graphene will reduce the resistance of the system, leading to a higher current response. This 517 proximity is achieved by the immobilizing dCas9 on the graphene, which then selectively binds target 518 DNA and keeps it close to the graphene surface. The dCas9 is bound to the graphene surface via a 519 PBA linker comprised of a planar pyrene ring system that electrostatically interacts with the 520 graphene and a carboxylate group that covalently couples to the dCas9 ( Figure 7A ). Subsequently the 521 rest of the surface is blocked to prevent the non-specific adsorption of charged molecules. A drop of 522 the sample is placed on the device, left to incubate, after which it is washed and the conductivity of 523 the system is measured via two platinum electrodes on the graphene. They report a LOD of 15 fM 524 and a total measurement time of 15 minutes (Hajian et al., 2019) . 525 The nanopore sensors exploit the binding of dCas9 effector proteins to DNA and are based on the 526 characteristic blockade signal of DNA-Cas9 complexes translocating through a nanopore ( Figure 7B) . 527 This translocation results in a dip in the ionic current through the pore that could be analyzed and 528 interpreted. Using nanopores as a sensing strategy enables detection of dsDNA without unzipping or 529 temperature changes and also opens up opportunities for multiplexing. Yang creating 'barcodes' in the ionic current traces specific for the different DNA sequences. In this way 534 they were able to identify two types of DNA target sequences in a mixture of 'background' DNA, 535 demonstrating the specificity and selectivity of the dCas9 effector protein in combination with 536 nanopore sensing. 537 While it is possible to observe the difference between the translocation signal of the dCas9 and the 538 DNA, the measurement method is still dependent on a single signal. While these means that a single 539 copy can be detected, it also means that for samples with low amounts of the specific DNA sequence, 540 the time needed for a measurement is very long, as the chance of the target DNA strand appearing in 541 the proximity of the pore is not very high. While nanopore sensing could be useful for identifying 542 DNA, it is low throughput, and therefore a very slow method for low LOD measurements. measuring directly at the cleavage site ( Figure 7C ). In a second system, a catalyst is placed on the 550 surface, while the compound to be oxidized is provided in the solution, which is then measured with 551 an electrode located at some distance in their device ( Figure 7D ). Another method uses an 552 electrochemical reaction with Ru(phen) 2 (dppz) 2+ -DNA complexes to create a red luminescence 553 indicating the presence of cleaved RNA after amplification. 554 There are systems using MB reported that are very closely related in their methods, with a few key 555 differences. Dai et al. exploited the trans-cleavage activity of Cas12a to obtain a high signal without 556 the need for DNA amplification via an electrochemical sensor (Dai et al., 2019) . In their "E-CRISPR" 557 sensor a gold measurement electrode (measuring 33 by 8 mm) is used, which is functionalized with a 558 thiolated-ssDNA-MB conjugate ( Figure 7C ). For their measurement, a (10 min preincubated) 20-30 µL 559 drop of sample is deposited onto the electrode area. In case of the target DNA being present, the 560 Cas12a complex is activated and will cut the single stranded DNA immobilized on the surface, which 561 releases the MB from the surface. After washing, a square wave voltammetry measurement is 562 performed, in which the reduction of MB will cause a peak in the current at a certain potential. The 563 removal of MB from the surface via cleavage decreases the current peak after the incubation step as 564 compared to before. This current difference indicates the presence of the target DNA and an 565 experimental LOD of 50 pM was obtained (Dai et al., 2019) . 566 DNA via proximity of an electrochemical signaling tag to a gold surface (Fan et al., 2003) . In this 569 sensor, they used single stranded hairpin DNA with a MB electrochemical signaling tag that was 570 immobilized on gold electrodes. In the standard sensor, without CRISPR-enhancement, the distance 571 between the signaling tag and electrode upon binding of target ssDNA to the hairpin will result in an 572 increase of the electron tunneling distance via the unfolding of the hairpin, measurable as a decrease 573 in the current. The addition of the Cas9 effector protein will cause the electrochemical signaling 574 probe to be released from the surface via cleavage upon recognition of PAM and target sequence 575 ( figure 7C-III) . This specific cleavage of Cas9 lowers the LOD of the "normal" sensor by at least two 576 orders of magnitude to 100 fM concentration of target DNA (Xu et al., 2020) . In this second 577 publication, the sensor was not (yet) incorporated in a POC device, but can be applied to a similar 578 electrode as the system of Dai et al. Using Cas12a, a larger signal was obtained and the LOD could be 579 reduced to 10 fM. The increase in sensitivity compared to the first sensor is caused using a DNA-580 hairpin, which brings the MB closer to the surface and increases the current response. However, the 581 fact that there is no collateral cleavage, but only cutting of the target DNA, means that another step 582 could be made towards increasing the sensitivity of the system. 583 The presence of the miRNA which induces collateral cleavage of an EXPAR specific pre-trigger 609 primer. In its uncleaved state the pre-trigger primer is not able to bind an EXPAR template, 610 however upon trans-cleavage by the RNP, the primer can hybridize with the amplification 611 template, initiating the formation of large amounts of dsDNA via an EXPAR amplification step. This 612 dsDNA formation, which acts as an signal amplification step, can be visualized by addition of 613 Ru(phen) 2 (dppz) 2+ , which produces a luminescence signal upon interaction by the major groove of 614 dsDNA. Under optimized conditions, a limit of detection down to 1 fM could be reached. Their 615 sensing method was translated to a paper-based bipolar electrode electrochemiluminescence 616 (pBPE-ECL) biosensing platform, consisting of a simple paper based device, on which an 617 electrochemical reaction was driven by a portable DC voltage supply. Their luminescence approach 618 makes specific detection possible without the need of extensive washing procedures, which 619 improves reproducibility and simplifies the experimental procedure, which is of great interest in 620 POC sensing (T. Zhou et al., 2020). applied to two possible systems for electronic readout. 625 The first system was a DNA-gel fuse. A conductive carbon black nanoparticle loaded DNA-gel was 626 cured on top of two interdigitated electrodes to connect them. If the DNA is cut by the RNP-target 627 DNA complex, the connection between the electrodes will be interrupted, increasing the resistance 628 of the system (English et al., 2019) . The gel acts as a fuse between the electrodes. For 629 measurements, the electrode pad is placed in an Eppendorf tube which also contains the solution 630 with the RNP complex. The measurement is however not quantitative and takes 3 to 12 hours, 631 depending on the concentration of target DNA (English et al., 2019) . To obtain a faster and more 632 quantitative result, the authors created a paper-based POC device, in which they combined optical 633 and electronic read-out (English et al., 2019) . They inserted a DNA cross-linked polyacrylamide gel 634 precursor, that together with ssDNA crosslinks to form a hydrogel gel in the paper channels. The 635 extent of gel formation depends inversely on the extent of degradation of the ssDNA in the gel. By 636 degrading the crosslinker via trans-cleavage by Cas12a, the channel is less obstructed by the gel, and 637 the level of flow of a colored buffer that was added afterwards can therefore be linked to the 638 amount of dsDNA that complemented with the Cas12a effector protein ( Figure 7E ). With the visual 639 output, a concentration down to 400 pM could be detected (English et al., 2019) . To reduce the user 640 error from the visual output, an electric readout was achieved by including two electrodes that 641 sandwiched the channel. These electrodes recorded the conductance, which depends on the number 642 of electrolyte ions in the channel introduced by the buffer. The conductance change could be directly 643 linked to the penetration length of the buffer and with this method a similar LOD could be obtained. 644 This device needs a pre-incubation time of 4 hours and a 2-minute readout. 645 646 The sequences one can detect using CRISPR/Cas effector proteins are limited, which is a general 7 problem in CRISPR sensing. CRISPR effector proteins are guided by crRNA and are able to recognize 8 target sequences on DNA and RNA that are complementary to the crRNA. The spacer sequence, 9 present on the crRNA, can be tailored to the target sequence. However, as discussed above for 10 (d)Cas9 and Cas12a/b, an effector specific PAM sequence is crucial to unwind the dsDNA strands and 11 allow complementation between crRNA and the target sequence. Depending on the Cas effector 12 protein family, mismatches between spacer and target sequence are tolerated. In general, the overall 13 mismatch tolerance depends on the number of mismatches and the position of the mismatch 14 compared to the PAM sequence, where distant mismatches are generally more tolerated. It was 15 shown for Cas9 that less than 5% of crRNA sequences with two or more mismatches to the target 16 sequence is functional in target binding (Anderson et al., 2015) and mismatches near the PAM region 17 are less tolerated (Pattanayak et al., 2013) . These limitations can generally be 47 overcome by shifting the target sequence in such a way that the first base following the protospacer 48 will be any base except a guanine (Gootenberg et al., 2018) . 49 Standardization is the key to effective POC CRISPR sensing. Both procedures and individual tests have 51 to be standardized in order to be sure that every user will obtain the same results. While 52 standardization is a general issue in POC testing, it becomes more important in the case of protein-53 based sensors. Salt levels, temperature, pH and/or the abundance of reaction inhibitors might 54 interfere in the assembly between (s)gRNA and effector protein and the cleavage activity of the 55 effector proteins, inducing a change in the generated signal ( 105 To lower the LOD, many of the published CRISPR biosensing methods require nucleic acid 106 amplification prior to CRISPR sensing. Standard amplification methods, such as PCR, are not suitable 107 for incorporation in POC devices due to the need for a bulky thermocycler in combination with the 108 cycling of multiple temperature steps from 60°C -95°C which typically leads to long reaction times 109 (≥90 minutes). 110 To allow CRISPR sensing in remote areas without trained personnel, multiple techniques based on 111 isothermal amplification can be used that are more straightforward to use and can be incorporated 112 in small handheld devices. Table 3 shows an overview of the isothermal amplification techniques that 113 have been applied in combination with CRISPR sensing. 114 The most often employed isothermal amplification methods are RPA and LAMP, which operate 115 between 37-42 °C (could also be used at room temperature, although this influences the reaction 116 kinetics), and ~65°C respectively. RPA and LAMP offer fast amplification times (from ~15 -60 min for 117 LAMP and 5-60 min for RPA depending on the initial nucleotide concentrations) which is of great 118 interest for POC applications. A major advantage of RPA over LAMP is that it requires much lower 119 temperatures during amplification. However, lower operation temperatures also have drawbacks, 120 such as non-specific amplification products and the formation of primer dimers (Zaghloul and El-121 Shahat, 2014). By the introduction of CRISPR-complexes, which are highly specific to their target 122 sequence, these unwanted products do not interfere with the sensing process and therefore do not 123 influence the end result. 124 When combining isothermal amplification and CRISPR sensing in a single POC device, the operation 127 temperature of the isothermal amplification should be in the same range as the operation 128 temperature of the RNP complex. Different effector proteins have different temperature optima. The 129 activity of Cas12a for example has an optimum temperature around 28°C, while Cas12b has an 130 optimal activity around 48°C. The high temperature tolerance of Cas12b was exploited in HOLMESv2, 131 where both LAMP and CRISPR sensing was incorporated in a one-pot reaction at 55°C (L. Li In their dynamic aqueous multiphase reaction (DAMR) system they took advantage of density 145 differences of sucrose concentrations . By phase separation, a two-phase system was 146 established. In the high-density bottom layer, the RPA reaction was initiated. The amplified 147 nucleotides could diffuse to the low-density top-phase where the Cas12a RNP could complement and 148 initiate collateral cleavage activity of quenched fluorescent probe sequences. In this way 149 incompatible reactions could be performed in one pot. The system was evaluated in reaction tubes 150 from (Wu et al., 2020b)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a)(Wu et al., 2020a) A and multiplex three-dimensional printed inserts for multi-well plates. It was found that 10-100 copies 151 of Human Papilloma Virus DNA spiked in untreated human plasma could be sensed in less than one 152 hour, showing the high tolerance of the DAMR system to inhibitors. 153 5 Conclusion and future outlook 154 While many CRISPR sensing applications were published in the last four years, the CRISPR nucleic acid 155 detection field is still very much in its infancy. As discussed in this review paper, there are still many 156 challenges before CRISPR sensing can replace established biomolecular nucleic acid sensing 157 techniques such as PCR. 158 In this review, we specifically focused on the POC detection of nucleic acids. It was shown that 159 CRISPR sensing may provide rapid diagnostics, but assay times are still influenced by the need for 160 target amplification. Therefore, type V and VI effector proteins have the highest potential in POC 161 applications as they provide signal amplification by the untargeted collateral cleavage activity of 162 reporter nucleic acids. This method has been used for fluorescent, colorimetric and electronic read- testing devices are needed, such as those based on microchips. The focus will mainly be on multiplex 177 sensing, allowing on-board quality checks and calibration, while measuring for multiple sequences 178 simultaneously. Furthermore, we expect a trend with (isothermal) amplification being replaced by 179 (genetically modified) Cas effector proteins with collateral cleavage activity for signal amplification 180 purposes. 181 The Weijerhorst Foundation is acknowledged for financial support. 183 RDMS is a minority stockholder of Self-screen B.V., a spin-off company of VUmc. 185 RNA 188 targeting with CRISPR-Cas13 C2c2 is a single-component programmable RNA-guided RNA-192 targeting CRISPR effector C2c2 is a single-component programmable RNA-guided RNA-197 targeting CRISPR effector. Science (80-. ). 353, aaf5573 Nucleic Acid Detection Using CRISPR/Cas Bio-sensing 200 Systematic analysis of CRISPR-Cas9 203 mismatch tolerance reveals low levels of off-target activity Cas12a-Based On-Site and Rapid Nucleic Acid Detection of African Swine Fever Clustered regularly interspaced short 209 palindrome repeats (CRISPRs) have spacers of extrachromosomal origin CRISPR-Cas12-based detection of SARS-214 CRISPR/Cas13a-Powered Electrochemical Microfluidic Biosensor for Nucleic Acid Amplification-217 Colorimetric detection of 219 nucleic acid sequences in plant pathogens based on CRISPR/Cas9 triggered signal amplification Visual detection of porcine 222 reproductive and respiratory syndrome virus using CRISPR-Cas13a The chemistry of Cas9 and its CRISPR colleagues CRISPR-227 Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science (80-. ) Use of up-230 converting phosphor reporters in lateral-flow assays to detect specific nucleic acid sequences: A 231 rapid, sensitive DNA test to identify human papillomavirus type 16 infection Multiplexed Recombinase Polymerase Amplification Assay to Detect Intestinal Protozoa An ultrasensitive, rapid, and portable coronavirus 237 SARS-CoV-2 sequence detection method based on CRISPR-Cas12 Exploring the Trans-240 Cleavage Activity of CRISPR-Cas12a (cpf1) for the Development of a Universal Electrochemical Chemie -Int Multiplexed Point-of-Care Testing -243 xPOCT All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for 245 Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus 248 Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. 249 Programmable CRISPR-responsive smart materials. Science (80-. ) Electrochemical interrogation of conformational changes as 254 a reagentless method for the sequence-specific detection of DNA Multiplexed 257 and portable nucleic acid detection platform with Cas13, Cas12a and Csm6. Science (80-. ) Nucleic acid detection with 262 CRISPR-Cas13a/C2c2. Science (80-. ) Toehold switches: De-novo-designed regulators of 264 gene expression A facile, rapid and 266 sensitive detection of MRSA using a CRISPR-mediated DNA FISH method, antibody-like 267 dCas9/sgRNA complex SARS-CoV-2 detection with 271 CRISPR SARS-CoV-2 detection with CRISPR 274 diagnostics Detection of unamplified target genes via CRISPR-Cas9 immobilized on a graphene field-278 effect transistor Programmed DNA destruction by miniature CRISPR-281 Cas14 enzymes Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System for Rapid Viral DNA Sensing High-286 throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-287 Cas12a and fluorescence based point-of-care system DNA targeting specificity of 291 RNA-guided Cas9 nucleases Single-Step, Salt-Aging-Free, and 293 Thiol-Free Freezing Construction of AuNP-Based Bioprobes for Advancing Nucleotide sequence of the 296 iap gene, responsible for alkaline phosphatase isoenzyme conversion in Escherichia coli, and 297 identification of the gene product A programmable 300 dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science (80-. ) A CRISPR-based assay for the 304 detection of opportunistic infections post-transplantation and for the monitoring of transplant 305 rejection PAM recognition by miniature CRISPR-Cas14 triggers programmable double-308 stranded DNA cleavage Challenges and perspectives in the development 310 of paper-based lateral flow assays A low-cost fluorescence reader 314 for in vitro transcription and nucleic acid detection with Cas13a SHERLOCK: nucleic acid 317 detection with CRISPR nucleases CRISPR-Cas13a mediated nanosystem for attomolar detection of canine parvovirus type 321 2 Genome-wide analysis reveals 323 specificities of Cpf1 endonucleases in human cells Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells Transcriptome 329 Engineering with RNA-Targeting Type VI-D CRISPR Effectors CRISPR/dCas9-mediated 332 biosensor for detection of tick-borne diseases CRISPR/dCas9-mediated 335 biosensor for detection of tick-borne diseases A guide to aid the selection of diagnostic tests. Bull. World 338 Health Organ Point-of-Care Testing: Principles, Practice, and Critical-340 Encyclopedia of Analytical Chemistry DCas9-mediated Nanoelectrokinetic Direct Detection of Target Gene for Liquid Biopsy The CCTL (Cpf1-assisted Cutting and Taq 346 DNA ligase-assisted Ligation) method for efficient editing of large DNA constructs in vitro HOLMESv2: A CRISPR-Cas12b-Assisted 349 Platform for Nucleic Acid Detection and DNA Methylation Quantitation Integrated Graphene Oxide Purification-Lateral Flow Test 352 Strips (iGOP-LFTS) for Direct Detection of PCR Products with Enhanced Sensitivity 355 CRISPR-Cas12a-assisted nucleic acid detection CRISPR/Cas Systems towards Next-Generation Biosensing CRISPR/Cas Multiplexed Biosensing: A Challenge or an Insurmountable 360 Obstacle? A CRISPR-Cas12a-derived biosensing platform for the highly sensitive 365 detection of diverse small molecules Evolutionary classification of 371 CRISPR-Cas systems: a burst of class 2 and derived variants Cas9 as a versatile tool for engineering biology Disposable nucleic acid biosensors based 376 on gold nanoparticle probes and lateral flow strip Challenges and 379 perspectives in the application of isothermal DNA amplification methods for food and water 380 analysis Cas9 effector complex assembly and the role of 3′-terminal segment of guide RNA Intervening sequences of 385 regularly spaced prokaryotic repeats derive from foreign genetic elements An ultrasensitive and specific point-of-care CRISPR/Cas12 based 389 lateral flow biosensor for the rapid detection of nucleic acids A high fidelity CRISPR/Cas12a based lateral flow biosensor for the detection of HPV16 393 and HPV18 Field-deployable viral diagnostics using CRISPR-Cas13. Science (80-. 399 ) Crystal structure of Cas9 in complex with guide RNA and target DNA Sequence-Specific Recognition of HIV-1 DNA with Solid-404 State CRISPR-Cas12a-Assisted Nanopores (SCAN) Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular 409 Components High-throughput profiling 411 of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity CRISPR elements in Yersinia pestis acquire new repeats 414 by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary 415 studies Adjacent Motif of Cas12a to Realize Visualized DNA Detection at the Single-Copy Level Free 418 from Contamination Dehydrated CRISPR-mediated DNA analysis for 421 visualized animal-borne virus sensing in the unprocessed blood sample. Sensors Actuators, B 422 Chem Rapid and Fully Microfluidic Ebola Virus Detection with CRISPR-425 Double nicking by RNA-guided CRISPR cas9 for enhanced 428 genome editing specificity Nucleic acid detection technologies and marker molecules in 430 bacterial diagnostics CRISPR-Cas12a Coupled with Platinum 432 Nanoreporter for Visual Quantification of SNVs on a Volumetric Bar-Chart Chip High-Resolution 436 Structure of Cas13b and Biochemical Characterization of RNA Targeting and Cleavage Recent advances in microfluidic sample preparation and 439 separation techniques for molecular biomarker analysis: A critical review Engineering of CRISPR-Cas12b for human genome editing Kinetic Basis for DNA 445 Target Specificity of CRISPR-Cas12a Detection Of White Spot Syndrome Virus In Shrimp RNA-451 Independent DNA Cleavage Activities of Cas9 and Cas12a CRISPR-Based Technologies: Impact of RNA-Targeting Systems A CRISPR Test for Detection of Circulating Nuclei Acids 458 Comparison of various repetitive DNA elements as genetic markers for strain differentiation and 459 epidemiology of Mycobacterium tuberculosis A lateral flow strip combined 462 with Cas9 nickase-triggered amplification reaction for dual food-borne pathogen detection Rolling Circular Amplification (RCA)-Assisted CRISPR/Cas9 Cleavage (RACE) for 466 Highly Specific Detection of Multiple Extracellular Vesicle MicroRNAs Clustered 469 Regularly Interspaced Short Palindromic Repeats/Cas9-Mediated Lateral Flow Nucleic Acid 470 Assay Multiplexed DNA Identification Using Site Specific dCas9 Barcodes and Nanopore Sensing The application of CRISPR-Cas for single species identification from 476 environmental DNA Contamination-free visual detection of CaMV35S 479 promoter amplicon using CRISPR/Cas12a coupled with a designed reaction vessel: Rapid, 480 specific and sensitive End-point dual 483 specific detection of nucleic acids using CRISPR/Cas12a based portable biosensor Regulated CRISPR-Cas12a Sensors for Point-of-Care Diagnostics of Non-Nucleic-Acid Targets Surpassing the detection limit and accuracy of the 489 electrochemical DNA sensor through the application of CRISPR Cas systems Fluorescent probe-based lateral flow assay for 492 multiplex nucleic acid detection Crystal Structure of Cpf1 in 496 Complex with Guide RNA and Target DNA Detection of CRISPR-dCas9 on DNA with Solid-State Nanopores Dynamic aqueous multiphase reaction 502 system for simple, sensitive and quantitative one-pot CRISPR-Cas12a based molecular diagnosis 503 Universal and Naked-Eye Gene Detection Platform Based on CRISPR/Cas12a/13a 507 System Recombinase polymerase amplification as a promising tool in 509 hepatitis C virus diagnosis Isothermal amplification methods for the detection of nucleic acids in 511 microfluidic devices A protocol for detection 516 of COVID-19 using CRISPR diagnostics The applications of CRISPR/Cas system in molecular 518 detection A sequence-specific plasmonic loop-mediated 520 isothermal amplification (LAMP) assay with orthogonal color readouts enabled by CRISPR 521 Cas12a CRISPR/Cas13a Powered Portable 523 Electrochemiluminescence Chip for Ultrasensitive and Specific MiRNA Detection A CRISPR-Cas9-triggered strand 526 displacement amplification method for ultrasensitive DNA detection Highlights • Rapid and reliable detection of nucleic acid is critical in many different fields • CRISPR/Cas effector protein complexes could be used for specific DNA detection without the need of complicated machinery and trained personnel in so-called point-of-care testing • A short and comprehensive introduction to CRISPR sensing is given • We review recent developments of integrating CRISPR sensing in Point-of-care devices • The challenges of (point-of-care) CRISPR sensing are discussed ☐ 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:RDMS is a minority stockholder of Self-screen B.V., a spin-off company of VUmc.