This study was supported in part by a Koch Institute Support Grant number P30-CA14051 from the National Cancer Institute (Swanson Biotechnology Center), a Core Center Grant P30-ES002109 from the National Institute of Environmental Health Sciences, the Koch Institute&#39;s Marble Center for Cancer Nanomedicine, the Koch Institute Frontier Research Program via the Kathy and Curt Marble Cancer Research Fund, and the Virginia and D. K. Ludwig Fund for Cancer Research. A.E.V.H. is supported by an NIH-funded predoctoral training fellowship (T32GM130546). S.N.B. is a Howard Hughes Medical Institute Investigator. L.H. is supported by a K99/R00 Pathway to Independence Award from the National Cancer Institute and the startup funding from Boston University.

All animal studies are approved by the Institutional Animal Care and Use Committee (IACUC) at the authors&#39; institution. Standard animal care facilities including housing chambers, sterile hoods, anesthetization, and ethical endpoint euthanization are required to properly carry out these experiments. All experiments are conducted in compliance with institutional and national guidelines and supervised by the veterinarian staff at the institution. Female BALB/c mice, used for the experiments, are obtained from a commerc...

CRISPR-Cas Synthetic Urine Biomarker Test for Cancer Diagnostics

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	<li>Soleimany, A. P., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-based+diagnostics:+an+emerging+paradigm+for+disease+detection+and+monitoring.">Activity-based diagnostics: an emerging paradigm for disease detection and monitoring.</a> Trends Mol Med. 26 (5), 450-468 (2020).</li><li>Muir, R. K., Guerra, M., Bogyo, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-based+diagnostics:+recent+advances+in+the+development+of+probes+for+use+with+diverse+detection+modalities.">Activity-based diagnostics: recent advances in the development of probes for use with diverse detection modalities.</a> ACS Chem Biol. 17 (2), 281-291 (2022).</li><li>Heitzer, E., Haque, I. S., Roberts, C. E. S., Speicher, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+and+future+perspectives+of+liquid+biopsies+in+genomics-driven+oncology.">Current and future perspectives of liquid biopsies in genomics-driven oncology.</a> Nat Rev Genet. 20 (2), 71-88 (2019).</li><li>Dudani, J. S., Warren, A. D., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+protease+activity+to+improve+cancer+care.">Harnessing protease activity to improve cancer care.</a> Annu Rev Canc Biol. 2, 353-376 (2018).</li><li>Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+new+dimensions.">Hallmarks of cancer: new dimensions.</a> Cancer Discov. 12 (1), 31-46 (2022).</li><li>Quail, D. F., Joyce, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+tumor+progression+and+metastasis.">Microenvironmental regulation of tumor progression and metastasis.</a> Nat Med. 19 (11), 1423-1437 (2013).</li><li>Kwong, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biomarkers:+a+twenty-first+century+path+to+early+cancer+detection.">Synthetic biomarkers: a twenty-first century path to early cancer detection.</a> Nat Rev Cancer. 21 (10), 655-668 (2021).</li><li>Kirkpatrick, J. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass-encoded+synthetic+biomarkers+for+multiplexed+urinary+monitoring+of+disease.">Mass-encoded synthetic biomarkers for multiplexed urinary monitoring of disease.</a> Nat Biotechnol. 31 (1), 63-70 (2013).</li><li>Chen, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas12a+target+binding+unleashes+indiscriminate+single-stranded+DNase+activity.">CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity.</a> Science. 360 (6387), 436-439 (2018).</li><li>Choi, H. 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Presented here is a highly customizable platform for multiplexed cancer detection with a portable urine test that assesses disease-associated proteolytic activity using a minimally invasive injected sensor. When activated by tumor proteases, peptide substrate cleavage is amplified via DNA barcode release into the urine. The synthetic DNA reporters in a urine sample can be read out by a secondary CRISPR-Cas-mediated enzymatic amplification using fluorometric detection or a simple paper-based test. DNA barcoding i...

Despite the significant effort to identify tumor biomarkers such as shed proteins and DNA, the cancer diagnostic field has been strained by their low abundance or rapid degradation in circulation1. As a complementary strategy, bioengineered synthetic biomarkers that selectively respond to disease features to generate amplified signals represent new avenues towards accurate and accessible diagnostics2,3. To aid detection, these synthetic biomarkers harness tumor-dependent activation mechanisms such as enzymatic amplification to produce analytes with improved signal-to-noise ratio4. Herein, a class of cancer-associated enzymes, proteases, are leveraged to activate injectable nanoscale sensors to release disease reporters detectable from the biofluids such as blood or urine5,6. In light of tumor heterogeneity, developing a panel of protease-activated sensors allows for multianalyte tests that combine different protease cleavage events into a &#39;disease signature&#39; to assess cancer incidence and progression in a more specific, multiplexed manner.
Protease-activated synthetic biomarkers have been developed that comprise peptide substrates conjugated to the surface of an inert carrier7. When injected in vivo, these peptides are carried to the tumor whereupon enzymatic cleavage by tumor proteases release reporters into blood or urine for detection. Multiplexed detection with protease-activated synthetic biomarkers requires each synthetic biomarker within a cocktail to be labeled with a unique molecular barcode. To this end, various approaches have been developed, including mass barcodes and ligand-encoded reporters8,9,10. As opposed to these methods of multiplexing which may be limited to a few different signal possibilities, DNA barcoding affords many more combinations in accordance with the high complexity and heterogeneity of human disease states. To expand the multiplexity of synthetic biomarkers, the sensors are barcoded by labeling each reporter with a unique DNA sequence for detection via CRISPR-Cas nuclease to amplify the biofluidic signal ex vivo. These single-stranded DNA (ssDNA) barcodes are designed to bind to complementary CRISPR guide RNAs (crRNAs), activating the target-triggered collateral nuclease activity of CRISPR-Cas12a11. This nuclease activity can be employed to cleave a reporter DNA strand detected through fluorescence kinetics or using paper strips.
In addition to molecular amplification via proteases (in vivo) and CRISPR-Cas (ex vivo), another key design feature of protease-activated synthetic biomarkers involves harnessing nanomaterial pharmacokinetics to increase diagnostic signal concentration in biofluids10. One approach is the use of a nanoparticle carrier to increase the circulation time of surface-conjugated peptide substrates. A polyethylene glycol (PEG) dendrimer is selected as a nanocarrier with relatively small hydrodynamic diameter and multivalency to increase delivery to tumors. While small enough to promote tumor delivery, the size of the PEG carrier is larger than the ~5 nm size cut-off of the kidney glomerular filtration barrier so that only cleaved peptide substrates can be cleared into the urine, taking advantage of size filtration by the kidneys12. In this protocol, the multiple-step workflow is outlined for the synthesis and application of DNA-barcoded activity-based nanosensors in a preclinical murine model, highlighting the setup of the CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test, which has been employed by this group to classify disease status in murine models of multiple cancer types13. Owing to the versatile design principle, all three functional components of the nanosensor - the nanocarrier (PEG polymer), the stimuli-responsive module (protease-activated substrate), and the biofluidic reporter (DNA barcode) - can be precisely interchanged according to application-specific needs, allowing for modularity by tailoring the target and release specificities.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x NEB Buffer 2.1</td>
			<td>New England Biolabs</td>
			<td>B6002SVIAL</td>
			<td></td>
		</tr>
		<tr>
			<td>20-mer phosphorothioated DNA reporters with 3&#8217;-DBCO group</td>
			<td>IDT</td>
			<td></td>
			<td>Custom DNA</td>
		</tr>
		<tr>
			<td>Agilent 1100 High Performance Liquid Chromatography system with Vydac 214TP510 C4 column</td>
			<td>&#160;Agilent</td>
			<td></td>
			<td>HPLC</td>
		</tr>
		<tr>
			<td>&#196;KTA fast protein liquid chromatography (FPLC)</td>
			<td>GE Healthcare</td>
			<td></td>
			<td>FPLC</td>
		</tr>
		<tr>
			<td>Amicon ultracentrifuge tubes (MWCO = 10 kDa)</td>
			<td>EMD millipore</td>
			<td></td>
			<td>Various volumes available</td>
		</tr>
		<tr>
			<td>Azide-terminated PAPs with C-terminus cysteine</td>
			<td>CPC Scientific</td>
			<td></td>
			<td>Custom peptide</td>
		</tr>
		<tr>
			<td>crRNAs&#160;</td>
			<td>IDT</td>
			<td></td>
			<td>See Supplementary Table 1</td>
		</tr>
		<tr>
			<td>Cryogenic transmission electron microscopy</td>
			<td>JEM-2100F</td>
			<td>JEOL</td>
			<td>cyroTEM</td>
		</tr>
		<tr>
			<td>Cysteine terminated DNA-peptide conjugates</td>
			<td>CPC Scientific</td>
			<td></td>
			<td>Custom peptide</td>
		</tr>
		<tr>
			<td>Dynamic light scattering (DLS)</td>
			<td></td>
			<td></td>
			<td>DLS</td>
		</tr>
		<tr>
			<td>EnGen LbaCas12a (Cpf1), 100 &#181;M</td>
			<td>New England Biolabs</td>
			<td>M0653T</td>
			<td></td>
		</tr>
		<tr>
			<td>Experimental animals</td>
			<td>Taconic Biosciences</td>
			<td>BALB/cAnNTac</td>
			<td>6&#8211;8&#8201;weeks of age</td>
		</tr>
		<tr>
			<td>gentleMACS C tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td>tissue homogenization</td>
		</tr>
		<tr>
			<td>HybriDetect Universal Lateral Flow Assay Kit</td>
			<td>Miltenyi Biotec</td>
			<td>MGHD 1</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix-assisted laser desorption/ionization&#8211;time of flight (MALDI&#8211;TOF) mass spectrometry&#160;</td>
			<td>Bruker</td>
			<td></td>
			<td>Microflex MALDI&#8211;TOF</td>
		</tr>
		<tr>
			<td>MC26-Fluc cell line</td>
			<td></td>
			<td></td>
			<td>Kenneth K. Tanabe Laboratory, Massachusetts General Hospital</td>
		</tr>
		<tr>
			<td>multivalent PEG (40 kDA, 8-arm) with maleimide-reactive group</td>
			<td>JenKem</td>
			<td>A10020-1 / 8ARM(TP)-MAL-40K,1 g</td>
			<td></td>
		</tr>
		<tr>
			<td>Python, Version 3.9</td>
			<td></td>
			<td></td>
			<td>https://www.python.org/</td>
		</tr>
		<tr>
			<td>Quant-iT OliGreen ssDNA Assay Kit and Quant-iT OliGreen ssDNA Reagent</td>
			<td>Invitrogen</td>
			<td>O11492</td>
			<td>ssDNA assay kit</td>
		</tr>
		<tr>
			<td>ssDNA FAM-T10-Quencher and&#160; FAM-T10-Biotin reporter substrates</td>
			<td>IDT</td>
			<td></td>
			<td>Custom DNA</td>
		</tr>
		<tr>
			<td>Superdex 200 Increase 10/300 GL column</td>
			<td>GE Healthcare</td>
			<td>GE28-9909-44</td>
			<td>For FPLC</td>
		</tr>
		<tr>
			<td>Tecan Infinite Pro M200 plate reader</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoFisher Pierce BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
	</tbody>
</table>

crispr-cas-mediated multianalyte synthetic urine biomarker test for portable diagnostics

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

Author Spotlight: Engineering Molecular Tools for Disease Detection and Imaging

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics

The Heart Lab is engineering molecular and cellular tools to precisely track and control disease biology. We use special information about diseased tissues to inform the design of responsive detection, imaging, and treatment tools for heterogeneous disorders. Biofluids present a convenient opportunity for disease detection and assessment, but these are often limited by low quantities of relevant analytes.As a result, much of the field is pursuing strategies to amplify disease associated signals to improve the sensitivity of diagnostic tests, like BCR and surface plasma on resonance. Rather than relying on endogenous disease biomarkers in circulation, our approach uses synthetic biomarkers to report disease activity when released from injected nanosensors. Our synthetic biomarkers are chemically stabilized DNA barcodes, which allow for multiple disease microenvironment signals to be detected at once, and identified using the corresponding barcode.

Nominating protease-activated peptide substrates 
To design sensors which will reflect changes in the proteolytic activity of the tissue, protease activity in the tissue is first characterized using a library of peptide probes13 (Figure 1). Fresh and frozen tissue samples can provide substantial information about the proteolytic activity of the tumor microenvironment by combining tissue samples with FRET probes designed to detect substrate cleava...

Watch this Scientific Journal Video about CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics at JoVE.com

This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities.

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for ...

crispr-cas-mediated-multianalyte-synthetic-urine-biomarker-test-for

Research

JoVE Journal

Bioengineering

Nanosensor Characterization and CRISPR-Cas Mediated Urine Biomarker Test for Cancer Diagnostics

To begin, dissolve two milligrams of multivalent PEG with maleimide reactive group, and one milliliter of 100 millimolar phosphate buffer. Filter the solution through a 0.2 micrometer filter. Add the cysteine terminated DNA peptide conjugates to the solution.Use size exclusion chromatography to remove the unconjugated materials. Run the samples in PBS. Monitor the absorbance of the to detect DNA and peptides.Place the sample in the centrifugal filter tubes and concentrate the synthesized nanosensors by centrifugation according to the manufacturer's recommended speed. Use a 96-well plate as the base for a customized housing chamber. To collect the urine for baseline measurements, first place a mouse in the housing chamber.Apply gentle pressure on the restrained mouse's bladder to expel any remaining urine onto the plate. Pipette the collected urine into a 1.5 milliliter tube. Prepare 200 microliters of the nanosensor injection solution in sterile PBS.Intravenously inject 200 microliters of the solution into each mouse. After one hour of injection, collect the urine samples from the control and experimental mice. For fluorescence-based CRISPR detection of the DNA barcodes, centrifuge the urine samples at 800 G for five minutes at room temperature.Now combine the CRISPR reagents, then add the Cas12a enzyme before gently pipetting the reaction mixture up and down. Incubate the reaction mixture at 37 degrees Celsius for 30 minutes. Then run the reporter reaction using the FRET-based reporter DNA mixture.Add the mixture to a 384-well plate and immediately measure the fluorescence at 37 degrees Celsius every two minutes for three hours to monitor the cleavage kinetics. To perform paper-based CRISPR detection, run an alternative reporter reaction following the incubation of the CRISPR reaction using a FAM-Biotin labeled DNA reporter. After combining the reagents into a 96-well plate, cover it with aluminum foil, then place the plate in an incubator at 37 degrees Celsius for 30 minutes.Now add 80 microliters of PBS into a fresh well of a 96-well plate. Pipette 20 microliters of reporter reaction mixture into this well. Place a lateral flow paper strip to each well, and wait until the liquid reaches the top of the strip.The chemically modified single-stranded DNA demonstrated Cas12a activation relative to the unmodified double stranded DNA and single stranded DNA. Modified DNA molecules in unprocessed urine showed colorimetric readout on lateral flow paper strips. The leading sample band indicated the release of detectable FAM through reporter cleavage triggered by urinary DNA.

584 Views.  Boston University.  This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities.

CRISPR-Cas Synthetic Urine Biomarker Test for Cancer Diagnostics

Summary