Integrating CRISPR with SERS: Toward intelligent point-of-care diagnostics of the future.

Chapter 9 - Selective Detection of Proteins and Nucleic Acids with Biofunctionalized SERS Labels

Molecular diagnostics, in particular, nucleic acid testing, is critical in a wide range of fields relevant to the quality of human life. Over the past few decades, quantitative polymerase chain reaction (qPCR) has remained the gold-standard technique for nucleic acid testing. However, qPCR is limited by expensive instruments, the need for operation by well-trained personnel, and long sample-to-results time. Recently, the discovery of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) systems has opened a new path for nucleic acid detection. Despite many advances, the current CRISPR-based biosensors in nucleic acid detection still require target preamplifications. The target amplification process often requires a set of proteins and primers (2-6) and additional sample preparation steps, which dramatically complicates and lengthens the detection process (20 min to 2 hours) as well as increases the contamination risk of patient samples. In addition, the target preamplification process is usually nonlinear and reaches saturation rapidly, limiting the ability to quantitatively measure viral copy numbers for predicting and/or monitoring disease progression. In this presentation, I will present a biosensor, termed CRISPR Cas13a-gFET, for target amplification-free nucleic acid detection with an attomolar sensitivity via harnessing the trans-cleavage mechanism of CRISPR Cas13a and ultrasensitive graphene field-effect transistor (gFET) (Angew. Chem. Int. Ed. 2022, 61(32): e202203826). This CRISPR Cas13a-gFET platform can be adapted to detect a variety of DNA targets for medical diagnostics, environmental monitoring, and food safety (ACS Sensors2023, 8, 4, 1489–1499). I will also present a point-of-care microfluidic device that offers ultrasensitive yet rapid detection of viral RNA from clinical samples (Lab on a Chip 2023, 23, 3862-3873). These CRISPR-based molecular diagnostic platforms significantly advanced our knowledge on the design and development of next-generation biosensors for target amplification-free detection and can be used for ultrasensitive detection of a wide range of nucleic acid targets with clinical, biological, and environmental importance.

Uncovering strong π-metal interactions on Ag and Au nanosurfaces under ambient conditions via in-situ surface-enhanced Raman spectroscopy

CRISPR-based adaptive and heritable immunity in prokaryotes

Ultrasensitive detection of nucleic acid with a CRISPR/Cas12a empowered electrochemical sensor based on antimonene

CRISPR: The frontier technology of next-generation RNA detection

Nucleic acids (deoxyribonucleic acid – DNA and ribonucleic acid – RNA) are essential components of all living organisms, with DNA encoding genetic information and RNA facilitating vital biological processes. The detection of nucleic acids having a specific sequence is crucial for identifying organisms and diagnosing genetic diseases. Because surface-enhanced Raman spectroscopy (SERS) is considered as one of the most promising analytical methods that offers important benefits such as short analysis time and exceptional sensitivity compared to other techniques, many groups are trying to apply SERS for nucleic acid detection. This review discusses how SERS spectroscopy can be used for DNA/RNA detection. Beginning with an overview of SERS theory, we delve into various SERS DNA/RNA sensors, including those based on a direct analysis of the SERS spectra of nucleic acids, and many types of sensors based on a selective hybridisation of probe and target nucleic acids. We describe how various types of sensors with increased sensitivity and reliability have evolved (from the first SERS DNA/RNA sensors described in the literature to recently developed ones). Challenges and future directions in SERS sensor development for nucleic acid detection and determination are also discussed. This comprehensive review aims to help researchers understand the field’s nuances, and to foster advancements in the use of SERS spectroscopy in the medical sector.

The nucleic acid detection using CRISPR/Cas biosensing system with micro-nano modality for point-of-care applications.

Surface-enhanced Raman scattering (SERS) has been recognized as a powerful tool for biosensors due to the ultrahigh sensitivity and unique fingerprint information. However, there are some limitations in trace target nucleic acid detection for the restricted signal-transducing and amplification strategies. Inspired by CRISPR/Cas12a with specific target DNA-activated collateral single-strand DNA (ssDNA) cleavage activity and liposome with signal molecule-loading properties, we first proposed a sensitive SERS-based on-site nucleic acid detection strategy mediated by CRISPR/Cas12a with trans-cleavage activity on ssDNA linkers utilized to capture liposomes. Liposomes loading two kinds of signal molecules, 4-nitrothiophenol (4-NTP) and cysteine, could achieve the dual-mode detection of target DNA with SERS and naked eye, respectively. The promptly amplified signals were initiated by the triggered breakdown of signal molecule-loaded liposomes. Emancipated 4-NTP, a biological-silent Raman reporter, would achieve highly selective and sensitive SERS measurement. Released cysteine induced the aggregation of plasmonic gold nanoparticles, leading to an obvious red to blue colorimetric shift to realize portable naked-eye detection. With this strategy, target nucleic acid concentration was dexterously converted into SERS and visualization signals and could be detected as low as 100 aM and 10 pM, respectively. The approach was also successfully applied to determine meat adulteration, achieving the detection of a low adulteration ratio in the complicated food matrix. We anticipate that this strategy will not only be regarded as a universal platform for the on-site detection of food authenticity but also broaden SERS application for the accurate determination of diverse biomarkers.

<p indent="0mm">Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR-Cas) systems have advanced rapidly for the detection of nucleic acids and molecular diagnoses. The sensitivity of techniques directly using CRISPR-Cas systems for target recognition and signal generation is limited by the kinetics of <italic>trans</italic>-cleavage. Thus, CRISPR-Cas systems have been coupled with isothermal amplification techniques. One strategy for integrating CRISPR-Cas and amplification reactions into a single-tube is to place reagents in separate locations within the tube, maintaining optimum conditions for each reaction. A more challenging strategy is to mix all reagents and allow nucleic acid amplification and CRISPR-based detection to proceed in a homogeneous solution. This desirable approach requires substantial understanding of the compatibility of enzymatic reactions, systematic optimization, and appropriate adjustments of the integrated reactions to ensure high sensitivity. Ultrasensitive techniques have been developed for the detection of SARS-CoV-2 in single-tubes. In this review, we highlight the principle, research needs, and challenges of ultrasensitive single-tube RNA detection using CRISPR technology. We stress the importance of understanding the kinetics of <italic>trans</italic>-cleavage activity of CRISPR-Cas systems.

The Broad Institute Scores Another Victory in Its Battle with the University of California over the Patenting of CRIPSR

Sample-to-answer nucleic acid detection using a fully integrated microdevice for nucleic acid extraction and smartphone-based droplet digital RPA/CRISPR.

Host-pathogen interactions are among the most prevalent and evolutionary important interactions known today. The predation of prokaryotes by their viruses is happening on an especially large scale and had a major influence on the evolutionary history of prokaryotes. Since most viruses are lytic at some point in their life-cycle, there is a high selection pressure for prokaryotes to develop defense mechanisms. As described in Chapter 1, the CRISPR-Cas system is a relatively recently discovered defense system and is also the first adaptive defense system discovered in prokaryotes. CRISPR-Cas systems are widespread, occurring in the majority of archaea and also a considerable fraction of bacteria. This diversity is also reflected in the diversity of different types of CRISPR-Cas systems, currently being divided into 6 major types with a large number of subtypes. The type I-E system of Escherichia coli is a well-studied model system and of high relevance, since it is a major subtype of type I systems which make up around 50 % of all discovered CRISPR-Cas systems. CRISPR-Cas systems basically comprise the CRISPR array, made up of repeats and foreign derived spacers, and a set of cas genes. Immunity is commonly divided into three functional stages, adaptation, expression and interference. Adaptation is the acquisition of new spacers from the foreign nucleic acid and its incorporation into the CRISPR array. During expression, the CRISPR array is transcribed, processed and assembled with Cas proteins into CRISPR RNA (crRNA) guided ribonucleoprotein complexes (crRNP). Interference is the detection, binding and destruction of foreign nucleic acids by the crRNP and in type I systems the Cas3 nuclease. The type I-E system contains another function, called primed adaptation. Primed adaptation is a more rapid and efficient version of regular (naïve) adaptation. In addition to the adaptation machinery, primed adaptation also requires the interference machinery. Chapter 2 describes and compares a fundamental feature of most, if not all, CRISPR-Cas systems and also many other small RNA based systems. The mode of action of small RNAs relies on protein-assisted base pairing of the guide RNA with target mRNA or DNA to interfere with their transcription, translation or replication. Several unrelated classes of small non-coding RNAs have been identified including eukaryotic RNA silencing associated small RNAs, prokaryotic small regulatory RNAs and prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) RNAs. All three groups identify their target sequence by base pairing after finding it in a pool of millions of other nucleotide sequences in the cell. In this complicated target search process, a region of 6 to 12 nucleotides of the small RNA termed the ‘seed’ plays a critical role. The seed is often a structurally pre-ordered region that increases accessibility and lowers the energy barrier of RNA-DNA duplex formation. Furthermore, the length of the seed is optimally chosen to allow rapid probing and also rejection of potential target sites. The seed is a perfect example of parallel evolution, showing that nature comes up with the same strategy independently multiple times. Chapter 3 provides a description and protocol of the Electrophoretic Mobility Shift Assay (EMSA) and its use for studying crRNPs. EMSA is a straightforward and inexpensive method for the determination and quantification of protein–nucleic acid interactions. It relies on the different mobility of free and protein-bound nucleic acid in a gel matrix during electrophoresis. Nucleic acid affinities of crRNPs can be quantified by calculating the dissociation constant (Kd ). Protocols for two types of EMSA assays are described using the Cascade ribonucleoprotein complex from Escherichia coli as an example. One protocol uses plasmid DNA as substrate, while the other uses short linear oligonucleotides. Plasmids can be easily visualized with traditional DNA staining, while oligos have to be radioactively labelled using the 32Phosphate isotope. The EMSA method and these protocols are applied throughout the other chapters of this thesis. Chapter 4 focusses on the processes of interference and primed adaptation, specifically on their tolerance of mutations. Invaders can escape Type I-E CRISPR-Cas immunity in E. coli by making point mutations in the protospacer (especially in the seed) or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive feedback process termed priming. Here, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6 nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of utilizing degenerate target regions with up to at least eleven mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position and is nucleotide dependent. Our findings imply that even out-dated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the co- evolutionary arms race with their invaders. In Chapter 5 we elucidate the mechanism of priming. Specifically, we determine how new spacers are produced and selected for integration into the CRISPR array during priming. We show that priming is directly dependent on interference. Rapid priming occurs when the rate of interference is high, delayed priming occurs when the rate of interference is low. Using in vitro assays and next generation sequencing, we show that Cas3 couples CRISPR interference to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step, PAM-associated manner. The helicase-nuclease Cas3 pre-processes target DNA into fragments of about 30–100 nt enriched for thymine-stretches in their 3’ ends. By reconstituting the spacer integration process in vitro, we show that the Cas1-2 complex further processes these fragments and integrates them sequence- specifically into CRISPR repeats by coupling of a 3’ cytosine of the fragment. Our results highlight that the selection of PAM-compliant spacers during priming is enhanced by the combined sequence specificities of Cas3 and the Cas1-2 complex, leading to an increased propensity of integrating functional CTT-containing spacers. In Chapter 6 we look deeper into a nucleotide specific effect on priming that was discovered in Chapter 4. Immunity is based on the complementarity of host encoded spacer sequences with protospacers on the foreign genetic element. The efficiency of both direct interference and primed acquisition depends on the degree of complementarity between spacer and protospacer. Previous studies focused on the amount and positions of mutations, not the identity of the substituted nucleotide. In Chapter 4, we describe a nucleotide bias, showing a positive effect on priming of C substitutions and a negative effect on priming of G substitutions in the basepairing strand of the protospacer. Here we show that these substitutions rather directly influence the efficiency of interference and therefore indirectly influence the efficiency of interference dependent priming. We show that G substitutions have a profoundly negative effect on interference, while C substitutions are readily tolerated when in the same positions. Furthermore, we show that this effect is based on strongly decreased binding of the effector complex Cascade to G mutants, while C mutants only minimally affect binding. In Chapter 5 we showed a connection between the rate of interference and the time of occurrence of priming. Here, we also quantify the extent of priming and show that priming is very prevalent in a population that shows intermediate levels of interference, while high or low levels of interference lead to a lower prevalence of priming. Chapter 7 describes an attempt to make use of our knowledge about the Cascade complex and develop it into a genome editing tool. The development of genome editing tools has made major leaps in the last decade. Recently, RNA guided endonucleases (RGENs) such as Cas9 or Cpf1 have revolutionized genome editing. These RGENs are the hallmark proteins of class II CRISPR-Cas systems. Here, we have explored the possibility to develop a new genome editing tool that makes use of the Cascade complex from E. coli. This RNA guided protein complex is fused to a FokI nuclease domain to sequence specifically cleave DNA. We validate the tool in vitro using purified protein and two sets of guide RNAs, showing specific cleavage activity. The tool requires two target sites of 32 nt each at a distance of 30-40 nt and inward facing three nucleotide flexible PAM sequences. Cleavage occurs in the middle between the two binding sites and primarily creates 4 nt overhangs. Furthermore, we show that an additional RFP can be fused to FokI-Cascade, allowing visualization of the complex in target cells. Unfortunately, we were not able to successfully apply the tool in vivo in eukaryotic cells.

Simple yet powerful clustered regularly-interspaced short palindromic repeats (CRISPR) technology has led to the advent of numerous developments in life sciences, biotechnology, therapeutics, and molecular diagnostics, enabled by gene editing capability. By exploiting the CRISPR-Cas system’s nucleic acid sequence detection abilities, CRISPR-based molecular diagnostics have been developed. Here, we review the development of rapid, sensitive, and inexpensive CRISPR-based molecular diagnostics. We introduce the transition of CRISPR technology to precision molecular diagnostic devices from tube to device. Next, we discuss the various nucleic acid (NA) detection methods by CRISPR. We address the importance of significant sample preparation steps for a future sample-to-answer solution, which is lacking in current CRISPR-based molecular diagnostic technology. Lastly, we discuss the extension of CRISPR-based molecular diagnostics to various critical applications. We envision CRISPR technology holds great promise for widespread use in precision NA detection applications after particular technical challenges are overcome.

Recent advancements in nucleic acid detection with microfluidic chip for molecular diagnostics