CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics
Summary
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.
Abstract
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.
Introduction
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 'disease signature' 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.
Protocol
Representative Results
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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'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.
Materials
| 10x NEB Buffer 2.1 | New England Biolabs | B6002SVIAL | |
| 20-mer phosphorothioated DNA reporters with 3’-DBCO group | IDT | Custom DNA | |
| Agilent 1100 High Performance Liquid Chromatography system with Vydac 214TP510 C4 column | Agilent | HPLC | |
| ÄKTA fast protein liquid chromatography (FPLC) | GE Healthcare | FPLC | |
| Amicon ultracentrifuge tubes (MWCO = 10 kDa) | EMD millipore | Various volumes available | |
| Azide-terminated PAPs with C-terminus cysteine | CPC Scientific | Custom peptide | |
| crRNAs | IDT | See Supplementary Table 1 | |
| Cryogenic transmission electron microscopy | JEM-2100F | JEOL | cyroTEM |
| Cysteine terminated DNA-peptide conjugates | CPC Scientific | Custom peptide | |
| Dynamic light scattering (DLS) | DLS | ||
| EnGen LbaCas12a (Cpf1), 100 µM | New England Biolabs | M0653T | |
| Experimental animals | Taconic Biosciences | BALB/cAnNTac | 6–8 weeks of age |
| gentleMACS C tubes | Miltenyi Biotec | 130-093-237 | tissue homogenization |
| HybriDetect Universal Lateral Flow Assay Kit | Miltenyi Biotec | MGHD 1 | |
| Matrix-assisted laser desorption/ionization–time of flight (MALDI–TOF) mass spectrometry | Bruker | Microflex MALDI–TOF | |
| MC26-Fluc cell line | Kenneth K. Tanabe Laboratory, Massachusetts General Hospital | ||
| multivalent PEG (40 kDA, 8-arm) with maleimide-reactive group | JenKem | A10020-1 / 8ARM(TP)-MAL-40K,1 g | |
| Python, Version 3.9 | https://www.python.org/ | ||
| Quant-iT OliGreen ssDNA Assay Kit and Quant-iT OliGreen ssDNA Reagent | Invitrogen | O11492 | ssDNA assay kit |
| ssDNA FAM-T10-Quencher and FAM-T10-Biotin reporter substrates | IDT | Custom DNA | |
| Superdex 200 Increase 10/300 GL column | GE Healthcare | GE28-9909-44 | For FPLC |
| Tecan Infinite Pro M200 plate reader | Tecan | ||
| ThermoFisher Pierce BCA Protein Assay Kit | ThermoFisher Scientific | 23225 |
Riferimenti
- Soleimany, A. P., Bhatia, S. N. Activity-based diagnostics: an emerging paradigm for disease detection and monitoring. Trends Mol Med. 26 (5), 450-468 (2020).
- Muir, R. K., Guerra, M., Bogyo, M. M. Activity-based diagnostics: recent advances in the development of probes for use with diverse detection modalities. ACS Chem Biol. 17 (2), 281-291 (2022).
- Heitzer, E., Haque, I. S., Roberts, C. E. S., Speicher, M. R. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nat Rev Genet. 20 (2), 71-88 (2019).
- Dudani, J. S., Warren, A. D., Bhatia, S. N. Harnessing protease activity to improve cancer care. Annu Rev Canc Biol. 2, 353-376 (2018).
- Hanahan, D. Hallmarks of cancer: new dimensions. Cancer Discov. 12 (1), 31-46 (2022).
- Quail, D. F., Joyce, J. A. Microenvironmental regulation of tumor progression and metastasis. Nat Med. 19 (11), 1423-1437 (2013).
- Kwong, G. A., et al. Synthetic biomarkers: a twenty-first century path to early cancer detection. Nat Rev Cancer. 21 (10), 655-668 (2021).
- Kirkpatrick, J. D., et al. Urinary detection of lung cancer in mice via noninvasive pulmonary protease profiling. Sci Transl Med. 12 (537), eaaw0262 (2020).
- Warren, A. D., et al. Disease detection by ultrasensitive quantification of microdosed synthetic urinary biomarkers. J Am Chem Soc. 136 (39), 13709-13714 (2014).
- Kwong, G. A., et al. Mass-encoded synthetic biomarkers for multiplexed urinary monitoring of disease. Nat Biotechnol. 31 (1), 63-70 (2013).
- Chen, J. S., et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 360 (6387), 436-439 (2018).
- Choi, H. S., et al. Renal clearance of quantum dots. Nat Biotechnol. 25 (10), 1165-1170 (2007).
- Hao, L., et al. CRISPR-Cas-amplified urinary biomarkers for multiplexed and portable cancer diagnostics. Nat Nanotechnol. 18 (7), 798-807 (2023).
- Hao, L., et al. Microenvironment-triggered multimodal precision diagnostics. Nat Mater. 20 (10), 1440-1448 (2021).
- Ghanta, K. S., et al. 5′-Modifications improve potency and efficacy of DNA donors for precision genome editing. Elife. 10, e72216 (2021).
- Bekkevold, C. M., Robertson, K. L., Reinhard, M. K., Battles, A. H., Rowland, N. E. Dehydration parameters and standards for laboratory mice. J Am Assoc Lab Anim Sci. 52 (3), 233-239 (2013).
