CRISPR-Cas Systems-Based Bacterial Detection

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1		Ismail Aziah	--	2183	2022-06-13 11:37:32	\|
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This entry is adapted from the peer-reviewed paper 10.3390/diagnostics12061335

Enzymes from clustered, regularly interspaced, short palindromic repeats (CRISPR)- CRISPR associated protein (Cas) systems have been adapted for the specific, rapid, sensitive, and portable sensing of nucleic acids. The CRISPR–Cas system is composed of RNA-guided endonucleases, and it is an adaptive immune system that protects its hosts from bacteriophage predation and parasitism by other mobile genetic elements (MGEs).

bacterial infections CRISPR Cas enzymes detection

1. Introduction

Bacterial infection occurs when bacteria enter the body, multiply, and cause a reaction in the body. Many patients with suspected bacterial infections are given empiric antimicrobial medicine instead of proper treatment, which leads to an increase in antimicrobial resistance ^[1]. The ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacteriaceae) are the microorganisms that are primarily involved in the resistance process, emphasising their ability to “escape” from common antibacterial treatments ^[2]. Antibiotic resistance pathogens have emerged as a result of a lack of rapid diagnostic tests with high sensitivity and specificity.

The majority of clinical microbiology laboratories still use the culture method for the detection of most bacteria from clinical samples; however, this process takes days to weeks to complete, relies on phenotypic biochemical characterization and requires skilled laboratory staff ^[3]^[4]. Apart from that, antibody detection methods have been used to detect circulating antibodies that are specific to respective bacteria; however, during acute infection, these results may be negative because the patients have not yet generated antibody response, and cross-reactions with unrelated IgM can occur ^[5]. Indeed, over the last two decades, there has been a surge in the development of diagnostic tests based on amplification and detection of specific bacterial nucleotide sequences. The majority of nucleic acid amplification methods use polymerase chain reaction (PCR) and can detect a pathogen of interest with high sensitivity and specificity; however, the requirement for expensive instruments (thermocycler) and reagents prevents such diagnostic tests from being used in areas with limited resources, such as on the battlefield or in developing countries ^[6]. As a result, the test cannot meet the ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Robust and rapid, Equipment-free, Deliverable) for developing point-of-care (POC) diagnostic tests ^[7].

Recently, enzymes from clustered, regularly interspaced, short palindromic repeats (CRISPR)- CRISPR associated protein (Cas) systems have been adapted for the specific, rapid, sensitive, and portable sensing of nucleic acids. The CRISPR–Cas system is composed of RNA-guided endonucleases, and it is an adaptive immune system that protects its hosts from bacteriophage predation and parasitism by other mobile genetic elements (MGEs) ^[8]. CRISPR-Cas system has been hailed as a versatile and reliable method for genome editing since its discovery. The CRISPR-Cas system has a diverse set of Cas proteins and genomic loci architecture, which has piqued researchers’ attention in a variety of biotech disciplines, including infectious disease detection ^[9]. Various CRISPR–Cas system-based methods have been developed for bacterial detection.

2. CRISPR-Cas Systems-Based Bacterial Detection

The CRISPR-Cas system has been used in various applications such as gene editing, identification of genotypes and SNPs, detection of antibiotics resistance and virulence genes, and diagnosis of infectious diseases ^[10]^[11]^[12]. Diagnostic techniques based on the CRISPR-Cas systems have recently attracted the attention of researchers due to their excellent accuracy. Three key aspects of the CRISPR-Cas systems contribute to high sensitivity and specificity in the diagnosis of disease, including the detection of bacteria. First, CRISPR– Cas systems identify specific amplicon sequences, distinguish them from amplification byproducts, and cut the sequences (cis-cleavage), a single-turnover method that improves specificity. Second, multiple turnover trans-cleavage activity of Cas12 and Cas13 causes nucleic acid signalling reporters to be cleaved several times, resulting in amplified readout signals for detection and hence improved sensitivity. Third, CRISPR–Cas systems make it easier to generate a variety of readout signals, which broadens their usefulness ^[13]^[14].

Bacteria from the genera Staphylococcus (17%), Escherichia (15%), Salmonella (13%), Listeria (8%), Mycobacterium (8%) and Streptococcus (8%) were mostly recognised by the CRISPR-Cas systems. Apart from that, the CRISPR-Cas systems were also utilised to detect bacteria from the genera (i) Vibrio (7%), (ii) Yersinia (3%), (iii) Pseudomonas (3%), (iv) Eberthella (3%), (v) Acinetobacter (3%), (vi) Bacillus (2%), (vii) Campylobacter (2%), (viii) Enterococcus (2%), (ix) Helicobacter (2%), (x) Klebsiella (2%), and (xi) Mycoplasma (2%). The majority of these bacteria are microbes responsible for the most common foodborne infections ^[15]. These pathogens are also antibiotic resistance bacteria and are on the World Health Organization (WHO)’s priority list for new antibiotic research and development ^[16]^[17]. A report by the United States Center for Disease Control and Prevention (CDC) provides an overview of the annual morbidity and mortality of antibiotic-resistant infections in the United States, estimating their number at approximately 2.8 million and the number of deaths associated with these infections at 35,000 ^[18].

The Cas enzyme is an endonuclease that may be programmed to detect DNA and RNA. CRISPR-Cas systems are divided into two classes (Class 1 and Class 2). Class 1 employs a multi-subunit crRNA-Cas protein, whereas Class 2 employs a single multidomain crRNA-Cas protein ^[19]. Class 2 is only found in bacteria, and accounts for less than 5% of all known systems. Each class has at least three types as well as several subtypes ^[20]. In Class 1, there are three types: I, III, and IV. Class 2 enzymes include the II (Cas9), V (Cas12), and VI (Cas13) enzyme classes, as well as subtypes such as V-A (Cas12a or Cpf1), V-B (Cas12b or C2c1), V-C (Cas12c or C2c3), V-F (Cas12f), VI-A (Cas13a or C2c2), VI-B (Cas13b or C2c4), VI-C (Cas13c or C2c7) and VI-D (Cas13d), which have evolved in separate evolutionary paths ^[21]^[22].

Cas12b, Cac12f, and dCas9 were utilised in a similar number of studies (4% each). Recently, researchers have shown interest in a new ortholog Cas12b (AapCas12b) because this enzyme (from Alicyclobacillus acidiphilus) can tolerate high temperature (60 °C) of isothermal reaction (e.g., LAMP) compared to Cas12a, which operates at a lower temperature (e.g., 25–40 °C), and so is incompatible with high-temperature conditions and leads to one-pot assays ^[23]^[24]. As well as AapCas12b, BrCas12b from (thermophile bacterium Brevibacillus sp. SYSU G02855) is also capable of binding and cleaving target DNA at high temperatures, making it a good candidate for diagnostic development ^[25]. Apart from Cas12b, Cas14a (Cas12f) is becoming more widely utilised in the diagnostic field because it demands full complementarity in the seed region of sgRNA, a trait that is important for obtaining single nucleotide specificity ^[26]. Cas12f, a type V effector protein, was previously known as Cas14. Cas14 is similar to Cas12 in that it can also target dsDNA and is dependent on T-rich PAM, hence it has been classed into the Cas12 family (Karvelis et al., 2020). Cas9 from Streptococcus pyogenes (SpCas9) is one of the simplest systems, drawing a lot of attention for its gene-editing capabilities ^[27]. However, in recent years, it has become a good bio-recognition element after being modified in a deactivated form (dCas9) resulting in an “antibody-like” mechanism. Researchers created a dCas9 by introducing two-point mutations, H840A and D10A, into the HNH and RuvC nuclease domains (dCas9). DNA cleavage activity is absent in dCas9, but DNA binding activity is unaffected ^[28].

Furthermore, Class 2 Cas enzyme selection is based on the enzymes’ properties. Cas9 (type II) has two nuclease domains, HNH and RuvC, which each cleaves one strand of double-stranded DNA (dsDNA) ^[29]. Cas12 (type V) has only one RuvC domain that cleaves dsDNA and single-stranded DNA (ssDNA) in the presence of cation ions such as magnesium and calcium ions (Mg²⁺ and Ca²⁺) ^[30]. Cas13 (type VI) has two predicted higher eukaryotic and prokaryotic nucleotide (HEPN) domains to cut single-stranded RNA (ssRNA) ^[31]. Cas9 requires both tracrRNA and crRNA, whereas Cas12 (except for Cas12b and Cas12c) and Cas13 utilise crRNA only. This is because Cas12 and Cas13 can cleave crRNA arrays to produce their crRNAs (self-processing) ^[31]^[32].

Cas9 recognise a specific PAM sequence (5′ NGG 3′) (N represents any nucleotide) in a non-target DNA strand, distant 10–12 nucleotides apart from the PAM sequence ^[29]. Cas12 identifies a 5′-T-rich PAM at the distal end. The PAM sequence required for Cas12 to bind dsDNA induces the catalytic activation of a crRNA-complementary dsDNA, but not of a crRNA-complementary ssDNA ^[33]. Cas13a identifies a 3′ end non-G protospacer flanking site (PFS) while 5′ end non-C PFS for Cas13b ^[34]^[35]. Moreover, Cas9 generate a blunt dsDNA break while Cas12 and Cas13 generate sticky and near U and A break respectively ^[30]^[36]. In addition, Cas9 does not exhibit trans-cleavage activity, but Cas12 and Cas13 do, allowing for powerful signal amplification ^[37]^[38]. These discoveries result in the development of the DETECTR (DNA endonuclease targeted CRISPR trans reporter) and SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) diagnostic platforms for Cas12 and Cas13 respectively ^[39]^[40]. Trans-cleavage activity by Cas12a and Cas13 were used in 83% of studies, while the remaining 17% of studies mostly used Cas9, which lacks trans-cleavage activity and hence used other signalling molecules.

Extracting bacterial genetic materials from samples necessitates determining the appropriate nucleic acid sequence (DNA or RNA) ^[41]. It is an important preanalytical stage in the development of any successful molecular diagnostic procedure, ensuring a reliable result. The extraction method was first used in 83% of studies before continuing to amplify and before CRISPR-Cas system-based detection. Extraction entails lysing the cells, purifying the nucleic acid to remove extraneous cell components, inhibiting compounds, degrading enzymes, and recovering the necessary nucleic acid ^[42]^[43]. The commercially available kits are also used for DNA extraction.

To improve sensitivity, the CRISPR-Cas system needs to be combined with a target nucleic acid amplification step performed by either PCR or isothermal technologies. This allows for the enrichment of rare and low-abundance nucleic acid targets and for the depletion of unwanted abundant nucleic acids. PCR is used in terms of targeted amplification of desired sequences, but it has several disadvantages, including the need for large equipment and trained personnel to operate it ^[44]. Isothermal amplification technologies address these constraints. CRISPR–Cas9 systems are effective for creating isothermal exponential amplification strategies because they can unwind dsDNA to ssDNA at a moderate temperature (37 °C) ^[13]. Isothermal methods were used in 66% of the investigations, while PCR methods were used in 23% of the studies. Among isothermal methods, RPA has been widely used in CRISPR-Cas system based bacterial detection followed by LAMP and SDA. RPA is notable for its ease of use, high sensitivity, selectivity, compatibility with multiplexing, exceptionally rapid amplification (20–60 min), and ability to operate at a low (37–42 °C) and constant temperature without the requirement for an initial denaturation phase or numerous primers ^[11]^[45]. The amplification technique, on the other hand, is not only time consuming but also poses a risk of aerosol contamination. Several research groups have worked hard to produce amplification-free CRISPR-Cas systems and investigate their use in pathogen identification. Only five (11%) of the 46 research studies found used an amplification-free CRISPR-Cas system-based bacterial detection.

Fluorescence detection (67%), lateral flow biosensor (13%), and electrochemical biosensor (11%) are three key detection approaches employed in CRISPR-Cas systems-based bacteria detection. Fluorescence-based sensing has several advantages, including background-free sensing, which dramatically improves the signal-to-noise ratio when compared to other optical approaches, but which necessitates the use of instruments (fluorescence reader, either portable or not, and a real-time PCR machine) ^[46]. The main benefit of LFAs is that their results are simple to interpret and can be read with the naked eye without the use of expensive instruments. LFAs, on the other hand, are inefficient and inaccurate, making these traditional paper-based platforms unsuitable for the higher quantitative analyses required in clinical applications ^[47]. Biosensors have several advantages over traditional analytical techniques, including excellent selectivity and sensitivity, the potential for miniaturization and portability, low cost, real-time detection, small sample volumes, and quick reaction ^[48].

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Ismail Aziah

Mohamad Ahmad Najib

Muhammad Fazli Khalid

Mehmet Ozsoz

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1. Introduction

2. CRISPR-Cas Systems-Based Bacterial Detection

References