CRISPR-Cas12a System Research Articles

PAM-independent ultra-specific activation of CRISPR-Cas12a via sticky-end dsDNA.

Although CRISPR-Cas12a [clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 12a] combiningpre-amplification technology has the advantage of high sensitivity in biosensing, its generality and specificity are insufficient, which greatly restrains its application range. Here, we discovered a new targeting substrate for LbaCas12a (Lachnospiraceae bacterium Cas12a), namely double-stranded DNA (dsDNA) with a sticky-end region (PAM-SE+ dsDNA). We discovered that CRISPR-Cas12a hadspecial enzymatic properties for this substrate DNA, including the ability to recognize and cleave it without needing a protospacer adjacent motif (PAM) sequence and a high sensitivity to single-base mismatches in that substrate. Further mechanism studies revealed that guide RNA (gRNA) formed a triple-stranded flap structure with the substrate dsDNA. We also discovered the property of low-temperature activation of CRISPR-Cas12a and, by coupling with the unique DNA hybridization kinetics at low temperature, we constructed a complete workflow for low-abundance point mutation detection in real samples, which was fast, convenient and free of single-stranded DNA (ssDNA) transformation. The detection limits were 0.005-0.01% for synthesized strands and 0.01-0.05% for plasmid genomic DNA, and the mutation abundances provided by our system for 28 clinical samples were in accordance with next-generation sequencing results. We believe that our work not only reveals novel information about the target recognition mechanism of the CRISPR-Cas12a system, but also greatly broadens its application scenarios.

Optimization of the technology for obtaining guide RNA using plate automatic synthesizer

A synthetic protocol was developed and optimal reagents have been selected for obtaining guide RNA oligonucleotides for the CRISPR/Cas system using ASM-2000 automatic synthesizer in 500 nmol scale. Methods for the isolation, purification and analytical control of synthetic RNA oligonucleotides have been developed. The improved technology has been used for preparation of guide RNAs for the CRISPR Cas12a system.

AND Logic-Gate-Based CRISPR/Cas12a Biosensing Platform for the Sensitive Colorimetric Detection of Dual miRNAs.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) system has been widely explored for the detection of disease-related nucleic acids. Nevertheless, the simultaneous detection of multiple nucleic acids within one assay using the CRISPR-Cas system is still challenging. In this study, we develop an AND logic-gate-based CRISPR-Cas12a biosensing platform to achieve the sensitive colorimetric detection of dual miRNAs. Specifically, the DNA probe was designed to recognize the binary input of miRNAs and to output trigger DNA, which activated the CRISPR-Cas12a system to cut single-stranded DNA (ssDNA). The ssDNA on magnetic beads (MBs) was cleaved by the activated CRISPR-Cas12a, causing the separation of glucose oxidase (GOx) from MB and the subsequent generation of a colorimetric signal. The color change induced by 1 pM of target miRNAs can be directly distinguished by the naked eye and the instrumental limit of detection reaches 36.4 fM. The overexpressed miR-205 and miR-944 in the real human serum can be detected, allowing us to differentiate between lung cancer patients and healthy people. Furthermore, the developed strategy achieves simultaneous detection of dual miRNAs using CRISPR-Cas12a with one kind of crRNA, avoiding sophisticated nucleic acid amplifications and the use of bulky instruments. The current method can broaden the CRISPR-Cas12a-based applications for multiple biomarkers detection and precise disease diagnosis.

MnO2 Nanozyme-Mediated CRISPR-Cas12a System for the Detection of SARS-CoV-2.

The CRISPR-Cas system was developed into a molecular diagnostic tool with high sensitivity, low cost, and high specificity in recent years. Colorimetric assays based on nanozymes offer an attractive point-of-care testing method for their low cost of use and user-friendly operation. Here, a MnO2 nanozyme-mediated CRISPR-Cas12a system was instituted to detect SARS-CoV-2. MnO2 nanorods linked to magnetic beads via a single-stranded DNA (ssDNA) linker used as an oxidase-like nanozyme inducing the color change of 3,3',5,5'-tetramethylbenzidine, which can be distinguished by the naked eye. The detection buffer color will change when the Cas12a is activated by SARS-CoV-2 and indiscriminately cleave the linker ssDNA. The detection limit was 10 copies per microliter and showed no cross-reaction with other coronaviruses. The nanozyme-mediated CRISPR-Cas12a system shows high selectivity and facile operation, with great potential for molecular diagnosis in point-of-care testing applications.

A one-pot RPA-CRISPR detection method for point-of-care testing of Enterocytozoon hepatopenaei infection in shrimp

The pathogen Enterocytozoon hepatopenaei (EHP) has significantly affected the shrimp farming industry since the last decade. For effective prevention, accurate and reliable detection methods for EHP infection are important, especially the methods suitable for point-of-care testing (POCT) environment. By combining recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system, this study developed a POCT-friendly detection method for EHP infection with one-pot procedure. The combined method utilized robust amplification capacity of RPA and high specificity of CRISPR, thus had better accuracy and reliability. The one-pot procedure was achieved by careful design and screening of the crRNA and systematic optimization of the reaction. The issues related to the compatibility of RPA and Cas12a cleavage have been successfully solved. The method was highly specific and the sensitivity reached 101 gene copies per reaction. The practical performance was validated with clinical shrimp samples. With the generous temperature requirement and convenient end-point reading of results, the one-pot method is highly suitable for POCT environment. It is a good example of one-pot RPA-CRISPR detection assay for POCT purpose, and has great application value in prevention of EHP infection in shrimp farming industry.

Genome-wide investigation of multiplexed CRISPR-Cas12a-mediated editing in rice.

Clustered regularly interspaced short palindromic repeats (CRISPR) nucleases like Cas9 and Cas12a are revolutionizing plant basic research and crop breeding. A major advantage of CRISPR over earlier nucleases systems is its capability of multiplexed genome editing. However, it remains unknown about the potential off-target effects when multiple concurrent DNA double-strand breaks (DSBs) are induced in a crop genome. Here, we investigated this important question in rice (Oryza sativa) using a highly multiplexed CRISPR-Cas12a system. With whole-genome sequencing, we first revealed high genome editing specificity of Mb2Cas12a and protospacer adjacent motif promiscuity of LbCas12a. We discovered large chromosomal rearrangement events in edited rice plants that endured many (e.g., >50) simultaneous DSBs, but not in plants that endured lower order DSBs (e.g., <10). Our results shed important light on the analysis and regulation of engineered crops derived from CRISPR-Cas mediated multiplexed genome editing.

Facile, ultrasensitive, and highly specific diagnosis of goose astrovirus via reverse transcription-enzymatic recombinase amplification coupled with a CRISPR-Cas12a system detection

Fatal gout in geese caused by goose astrovirus (GAstV) has been spreading rapidly in China since 2018, causing serious economic losses in the goose breeding industry. To achieve simple, convenient and sensitive detection of GAstV, a novel diagnostic test was developed by combining reverse transcription-enzymatic recombinase amplification (RT-ERA) and CRISPR-Cas12a technologies. RT-ERA primers were designed to pre-amplify the conserved region of the ORF2 gene of GAstV and the predefined target sequence detected using the Cas12a/crRNA complex at 37℃ for 30 min. Specific detection of GAstV was achieved with no cross-reaction with non-GAstV templates and a sensitivity detection limit of 2 copies. The experimental procedure could be completed within 1 h, including RNA extraction (15 min), RT-ERA reaction (20 min), CRISPR-Cas12a/crRNA detection (5 min) and result readout (within 2 min) steps. In conclusion, the combination of RT-ETA and CRISPR-Cas12a provides a rapid and specific method that should be effective for the control and surveillance of GAstV infections in farms from remote locations.

Novel CRISPR-based detection of Leishmania species.

Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods leads to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with Leishmania (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L. (Viannia) subgenus. Our CRISPR-based assays were capable of detecting down to 5 × 10−2 (kDNA) or 5 × 100 (18S rDNA) parasite genome equivalents/reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan-Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L. (Viannia) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L. (Viannia) subgenus levels.

A CRISPR-Cas12a-based diagnostic method for multiple genotypes of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) infection is commonly reported in countries of Northeast Asia including China, Japan and South Korea. The majority of the SFTS patients are elderly and the average fatality rate is more than 10%. A rapid and sensitive diagnostic method to monitor and prevent SFTSV transmission remains an urgent clinical challenge. In this study, we developed a molecular diagnostic technique for detection of SFTSV using the CRISPR-Cas12a system combined with reverse transcription recombinase polymerase amplification (RT-RPA). Using this method, we successfully diagnosed SFTSV infections with the reaction time of 50 min from blood plasma without cross-reactivity to other viruses, supporting its application for rapid and sensitive diagnosis of SFTS.

Diagnostics of Infections Produced by the Plant Viruses TMV, TEV, and PVX with CRISPR-Cas12 and CRISPR-Cas13.

Viral infections in plants threaten food security. Thus, simple and effective methods for virus detection are required to adopt early measures that can prevent virus spread. However, current methods based on the amplification of the viral genome by polymerase chain reaction (PCR) require laboratory conditions. Here, we exploited the CRISPR-Cas12a and CRISPR-Cas13a/d systems to detect three RNA viruses, namely, Tobacco mosaic virus, Tobacco etch virus, and Potato virus X, in Nicotiana benthamiana plants. We applied the CRISPR-Cas12a system to detect viral DNA amplicons generated by PCR or isothermal amplification, and we also performed a multiplexed detection in plants with mixed infections. In addition, we adapted the detection system to bypass the costly RNA purification step and to get a visible readout with lateral flow strips. Finally, we applied the CRISPR-Cas13a/d system to directly detect viral RNA, thereby avoiding the necessity of a preamplification step and obtaining a readout that scales with the viral load. These approaches allow for the performance of viral diagnostics within half an hour of leaf harvest and are hence potentially relevant for field-deployable applications.

The Off-Target Effect of CRISPR-Cas12a System toward Insertions and Deletions between Target DNA and crRNA Sequences.

The CRISPR-Cas12a system is a new type of genome editing tool with high efficiency and targeting. However, other sequences in the genome may also be cleaved nonspecifically, resulting in unavoidable off-target effects. Therefore, it is necessary to learn more about the mechanism of CRISPR-Cas12a to recognize target sequences to avoid its off-target effects. Here, we show that insertion (DNA bubble) or deletion (RNA bubble) of the target dsDNA sequence compared with the crRNA sequence, the CRISPR-Cas12a system can still recognize and cleave the target dsDNA sequence. We conclude that the tolerance of CRISPR-Cas12a to the bubbles is closely related to the location and size of the bubble and the GC base content of crRNA. In addition, we used the unique property of CRISPR-Cas12a to invent a new method to detect mutations and successfully detect the CD41-42(-CTTT) mutation. The detection limit of this method is 0.001%. Overall, our results strongly indicate that in addition to considering off-target effects caused by base mismatches, a comprehensive off-target analysis of the insertion and deletion of the target dsDNA sequence is required, and specific guidelines for effectively reducing potential off-target cleavage are proposed, to improve the safety manual of CRISPR-Cas12a biological application.

A CRISPR-Cas12a-powered magnetic relaxation switching biosensor for the sensitive detection of Salmonella

Magnetic relaxation switching (MRS) biosensors are attractive in the field of food safety owing to their simplicity and high signal-to-noise ratio. But they are less in sensitivity and stability caused by the insufficient crosslinking or non-specific binding of magnetic nanoparticles (MNPs) with targets. To address this problem, the CRISPR-Cas12a system was introduced into an MRS biosensor for the first time, to precisely control the binding of two types of MNPs with sizes of 130 nm (MNP130) and 30 nm (MNP30), for the sensitive detection of Salmonella. Delicately, the biosensor was designed based on the different magnetic properties of the two sizes of MNPs. The target Salmonella activated the collateral cleavage activity of the CRISPR-Cas12a system, which inhibited the binding of the two sizes of MNPs, resulting in an increase of unbound MNP30. After separating MNP130-MNP30 complexes and MNP130 from MNP30, the free MNP30 left in solution acted as transverse relaxation time (T2) signal reporters for Salmonella detection. Under optimized conditions, the CRISPR-MRS biosensor presented a limit of detection of 1.3 × 102 CFU mL−1 for Salmonella, which is lower than most MRS biosensor analogues. It also showed satisfactory specificity and performed well in spiked chicken meat samples. This biosensing strategy not only extends the reach of the CRISPR-Cas12a system in biosensors but also offers an alternative for pathogen detection with satisfactory sensitivity.

A novel “signal on-off-super on” sandwich-type aptamer sensor of CRISPR-Cas12a coupled voltage enrichment assay for VEGF detection

Vascular endothelial growth factor (VEGF) plays an important role in atherosclerosis, and the detection of VEGF is critical for the prevention, monitoring, and diagnosis of cardiovascular diseases. Here, a novel “signal on-off-super on” sandwich-type aptamer sensor with a triple signal amplification strategy was developed for the first time. Based on the capture aptamer was labeled with methylene blue (MB) on the internal bases, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-coupled voltage enrichment was used to amplify the electrochemical signal. To improve the analytical performance of the aptamer sensor, gold nanoparticles@Ti3C2Tx-Mxene (AuNPs@Ti3C2Tx-Mxene) were synthesized through the electrodeposition of AuNPs on the Ti3C2Tx-Mxene surface, providing active sites for the immobilization of the aptamer and amplifying the electrochemical signals. The excellent trans-cleavage activity of the CRISPR-Cas12a system was harnessed to cleave signal probes. The cleaved signal probes were enriched using an electrochemical signal instead of complicated target amplification steps before detection. Hence, we report a simplified detection process for amplifying electrochemical signals. Under optimal conditions, the aptamer sensor exhibited high sensitivity, acceptable stability, and reproducibility with a wide linear range from 1 pM to 10 μM (R2 = 0.9917) and an ultralow detection limit of 0.33 pM (S/N = 3). Therefore, we propose a novel strategy of CRISPR-Cas12a-based protein detection that opens a new window for the diagnostic applications of various biomarkers.

CRISPR-Cas12a-Derived Photoelectrochemical Biosensor for Point-Of-Care Diagnosis of Nucleic Acid

This work presented a point-of-care (POC) photoelectrochemical (PEC) biosensing for the detection of human papillomavirus-16 (HPV-16) on a portable electrochemical detection system by using CRISPR-Cas12a trans-cleaving the G-quadruplex for the biorecognition/amplification and a hollow In2O3-In2S3-modified screen-printed electrode (In2O3-In2S3/SPE) as the photoactive material. G-quadruplexes were capable of biocatalytic precipitation (H2O2-mediated 4-chloro-1-naphthol oxidation) on the In2O3-In2S3/SPE surface, resulting in a weakened photocurrent, but suffered from trans-cleavage when the CRISPR-Cas12a system specifically recognized the analyte. The photocurrent results could be directly observed with the card-sized electrochemical device via a smartphone, which displayed a high-value photocurrent for these positive samples, while a low-value photocurrent for the target-free samples. Such a system exhibited satisfying photocurrent responses toward HPV-16 within a wide working range from 5.0 to 5000 pM and allowed for detection of HPV-16 at a concentration as low as 1.2 pM. The proposed assay provided a smartphone signal readout to enable the rapid screening PEC determination of HPV-16 concentration without sophisticated instruments, thus meeting the requirements of remote areas and resource-limited settings. We envision that combining an efficient biometric PEC sensing platform with a wireless card-sized electrochemical device will enable high-throughput POC diagnostic analysis.

CRISPR-Cas12a combination to alleviate the false-positive in loop-mediated isothermal amplification-based diagnosis of Neisseria meningitidis

BackgroundLoop isothermal amplification (LAMP) has recently been proposed as a point-of-care diagnostic tool to detect acute infectious pathogens; however, this technique embeds risk of generating false-positive results. Whereas, with abilities to accurately recognize specific sequence, the CRISPR/Cas12a can forms complexes with cognate RNA sensors and cleave pathogen’s DNA targets complimerntary to its cognate RNA, afterward acquiring the collateral activity to unbiasedly cut nearby off-target fragments. Therefore, if relevant fluorescent-quencher-nucleic probes are present in the reaction, the non-specific cleavage of probes releases fluorescences and establish diagnostic read-outs.MethodsThe MetA gene of N. meningitidis was selected as target to optimize the LAMP reaction, whereas pseudo-dilution series of N. meningitidis gemonics DNA was used to establish the detection limit of LAMP/Cas12a combination assay. The diagnostic performance of established LAMP/Cas12a combination assay was validated in comparation with standard real-time PCR on 51 CSF samples (14 N. meningitidis confirmed patients and 37 control subjects).ResultsIn relevant biochemical conditions, CRISPR-Cas12a and LAMP can work synchronously to accurately identify genetics materials of Nesseria menitigistis at the level 40 copies/reaction less than 2 h.ConclusionsIn properly optimized conditions, the CRISPR-Cas12a system helps to alleviate false positive result hence enhancing the specificity of the LAMP assays.

Engineered Cas12a-Plus nuclease enables gene editing with enhanced activity and specificity

BackgroundThe CRISPR-Cas12a (formerly Cpf1) system is a versatile gene-editing tool with properties distinct from the broadly used Cas9 system. Features such as recognition of T-rich protospacer-adjacent motif (PAM) and generation of sticky breaks, as well as amenability for multiplex editing in a single crRNA and lower off-target nuclease activity, broaden the targeting scope of available tools and enable more accurate genome editing. However, the widespread use of the nuclease for gene editing, especially in clinical applications, is hindered by insufficient activity and specificity despite previous efforts to improve the system. Currently reported Cas12a variants achieve high activity with a compromise of specificity. Here, we used structure-guided protein engineering to improve both editing efficiency and targeting accuracy of Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a).ResultsWe created new AsCas12a variant termed “AsCas12a-Plus” with increased activity (1.5~2.0-fold improvement) and specificity (reducing off-targets from 29 to 23 and specificity index increased from 92% to 94% with 33 sgRNAs), and this property was retained in multiplex editing and transcriptional activation. When used to disrupt the oncogenic BRAFV600E mutant, AsCas12a-Plus showed less off-target activity while maintaining comparable editing efficiency and BRAFV600E cancer cell killing. By introducing the corresponding substitutions into LbCas12a, we also generated LbCas12a-Plus (activity improved ~1.1-fold and off-targets decreased from 20 to 12 while specificity index increased from 78% to 89% with 15 sgRNAs), suggesting this strategy may be generally applicable across Cas12a orthologs. We compared Cas12a-Plus, other variants described in this study, and the reported enCas12a-HF, enCas12a, and Cas12a-ultra, and found that Cas12a-Plus outperformed other variants with a good balance for enhanced activity and improved specificity.ConclusionsOur discoveries provide alternative AsCas12a and LbCas12a variants with high specificity and activity, which expand the gene-editing toolbox and can be more suitable for clinical applications.

Target recognition triggered CRISPR-Cas12a assisted allosteric scaffold for sensitively analyzing bacterial infection after dental implantation

Accurate identification and sensitive quantification of infected bacteria after dental implant both play crucial roles in early-diagnosis of bacterial infection and guiding medicine applications. Herein, we propose a sensitive and accurate bacteria detection method based on CRISPR-Cas12a system assisted allosteric scaffold. In the method, the allosteric scaffold takes the responsibility of specifically identifying target bacteria and inducing CRISPR-Cas12a based signal amplification. Eventually, the method exhibits a wide detection range from 6 × 106 cfu/mL to 6 × 102 cfu/mL with the limit of detection (LOD) of 47 cfu/mL. Furthermore, the established approach also possesses a high specificity due to high selectivity of aptamer and robust accuracy in recognizing double strand DNA by CRISPR-Cas12a system. We believe that this work can provide new strategies in the field of diagnosing bacterial infections after dental implantation.

Development of a RPA-CRISPR-Cas12a Assay for Rapid, Simple, and Sensitive Detection of Mycoplasma hominis.

Mycoplasma hominis, which is difficult to culture and identify by ordinary methods, is one of the smallest pathogens in the human genitourinary tract causing urogenital infections. A CRISPR-Cas12a-based detection system might provide a novel application for M. hominis nucleic acid detection in molecular diagnostics. A plasmid containing the glyceraldehyde-3-phosphate dehydrogenase gene of M. hominis (ATCC_27545) as the positive control was constructed by homologous recombination. The active Cas12a protein was purified by affinity chromatography. The primers for recombinase polymerase amplification (RPA), the CRISPR RNA (crRNA), and the ratio of Cas12a to crRNA were further optimized. Finally, the sensitivity, specificity, and clinical effectiveness of the Cas12a detection system were confirmed. We successfully constructed and optimized a novel nucleic acid detection system for M. hominis based on RPA-CRISPR-Cas12a, and the whole process takes only 1 h. The limit of detection for the gap gene of M. hominis was 3 copies/μl and no cross-reactivity with other urogenital pathogens appeared. In the evaluation of 111 clinical samples, the sensitivity and specificity were both 1.000 and the area under the curve of the receiver operating characteristic was 1.000 (p < 0.001), indicating that the RPA-Cas12a-fluorescent assay was fully comparable to the traditional culture method. Finally, the RPA-Cas12a detection system can also be combined with lateral flow strips (LFS) to achieve visual detection. We successfully developed a low-cost and rapid detection method of M. hominis based on RPA-Cas12a technology. This method realized by fluorescence value readout and visual detection by LFS could be applied in population screening and resource-limited conditions.

CRISPR-Cas12a based fluorescence assay for organophosphorus pesticides in agricultural products

Herein, we propose a sensitive fluorescent assay for organophosphorus pesticides (OPs) detection based on a novel strategy of activating the CRISPR-Cas12a system. Specifically, acetylcholinesterase (AChE) hydrolyzes acetylthiocholine into thiocholine (TCh). Subsequently, TCh induces the degradation of MnO2 nanosheets and generates sufficient Mn2+ ions to activate the Mn2+-dependent DNAzyme. Then, as the catalytic product of activated DNAzyme, the short DNA strand activates the CRISPR-Cas12a system to cleave the fluorophore-quencher-labeled DNA reporter (FQ) probe effectively; thus, increasing the fluorescence intensity (FI) in the solution. However, in the presence of OPs, the activity of AChE is suppressed, resulting in a decrease in FI. Under optimized conditions, the limits of detection for paraoxon, dichlorvos, and demeton were 270, 406, and 218 pg/mL, respectively. Benefiting from the outstanding MnO2 nanosheets properties and three rounds of enzymatic signal amplification, the proposed fluorescence assay holds great potential for the detection of OPs in agricultural products.

Hollow covalent organic framework-sheltering CRISPR/Cas12a as an in-vivo nanosensor for ATP imaging

In addition to applications in genome editing, clustered regularly interspaced short palindromic repeats (CRISPR) have recently been engineered for medical diagnostics based on their trans-cleavage activity owing to their high base resolution and isothermal signal amplification. However, trans-cleavage activity is too fragile to be applied in vivo. Herein, we introduce a hollow covalent organic framework (COF)-sheltering CRISPR/aptamer-based sensor (h-CCS) for ATP imaging in living animals. The CRISPR/aptamer-based complex is comprised of the CRISPR-Cas12a system, fluorophore quencher-labeled single-stranded DNA substrate (ssDNA-FQ), and a DNA activator that pre-hybridizes with ATP aptamer to prevent the trans-cleavage activity of the Cas12a system in the absence of ATP. After being encapsulated in a hollow COF, the constructed nanoreactor is highly robust and can be lit up by ATP for in vivo imaging. Considering the unique properties of h-CCS, this strategy offers great potential to broaden applications of not only CRISPR-Cas systems but also other proteins in porous matrixes for clinical diagnostics, medical research, and biomimetic nanodevices.