Processing
Although CRISPR technology is usually thought of as an approach to genome editing, it can also be used to diagnose a wide variety of diseases [1]. Many CRISPR enzymes, including Cas9 and the Cas12, Cas13, and Cas14 families have shown promise in diagnosis of infectious diseases (viruses, bacteria, etc.), cancer, and many other diseases that in some way involve a nucleic acid sequence (either RNA or DNA) [2].
Cas9 diagnostic methods
Several techniques that use Cas9 have been developed as diagnostic methods. Some of these methods use enzymatically active Cas9, while others use catalytically inactive (dead) Cas9 (dCas9).
Active Cas9-based systems
Methods that use catalytically active Cas9 for disease diagnosis can be divided into 2 groups: those that use Cas9 in a sample-preparation step, and those that use Cas9 in some aspect of a detection step.
Cas9 has been used for the preparation step of a diagnostic method known as FLASH (Finding Low Abundance Sequences by Hybridization) [3]. “Hybridization” in this context refers to the CRISPR RNA (crRNA) in a Cas9 ribonucleoprotein “hybridizing” to target DNA. In FLASH, Cas9 is targeted not to 1 site but to hundreds of sites using a large set of crRNAs. These crRNAs are designed to target Cas9 to break up a pathogen’s genomic DNA into pieces of the right size for next generation sequencing (NGS). Amplicon sequencing NGS is then performed to allow detection of the presence of the sequences of a large number of genes such as antibiotic resistance genes [3].
The NASBA-CRISPR cleavage (NASBACC) approach is an example of a system using catalytically active Cas9 in a detection step. This portable, low-cost methodology is designed to be usable in low-tech conditions [4]. It can distinguish American from African Zika virus strains, whose genomes differ by only 1 base in a protospacer-adjacent motif (PAM) site. This approach involves reverse transcription and isothermal amplification of RNA in a process known as nucleic acid sequence-based amplification (NASBA), which produces enough dsDNA to be cleaved by Cas9. Depending on the DNA sequence, the dsDNA will either be cleaved (if there is a PAM site) or will not be cleaved (if the PAM site is altered by a single base, as in the case of these 2 virus strains). During the NASBA reaction, not only is DNA amplified but a reporter gene (LacZ) is also expressed. This allows a change from yellow to purple on a paper detector. However, if the target sequence is present and therefore amplified, Cas9 cleaves the DNA, and there is no color change.
There are also several other Cas9-based detection methods, developed to diagnose human papilloma virus [5] and other diseases [1, 2, 6]. Cas9-based methods are highly generalizable, because using a different gRNA sequence directs Cas9 to any specified target site with an adjacent PAM. Therefore, in addition to the diseases these methods have been specifically shown to detect, these methods can be adapted to detection of many other diseases as well.
dCas9-based systems
The “PC” (which stands for “paired dCas9”) reporter system is an example of a system using dCas9. This technique consists of 2 molecules of dCas9 each connected to a half-molecule of firefly luciferase. When both dCas9 molecules are directed by guide RNA (gRNA) to pre-specified DNA sequences near each other in the genome of the pathogen of interest, the 2 halves of the luciferase come together, and light is emitted [7]. The PC reporter system is extremely sensitive, detecting as little as 1 molecule of DNA [1] and was shown to detect disease agents such as tuberculosis [7] and likely any DNA sequence, as long as there are 2 good target sequences at an appropriate distance from each other.
Another dCas9-based diagnostic system with a simpler approach can be used with unpurified bacterial whole-cell lysates [8]. Like many CRISPR-Cas9–based methods, no PCR or other amplification step is needed. This method targets His-tagged dCas9 to a specific site in pathogenic DNA and then uses a nickel-bead pull-down approach to immobilize dCas9 bound to DNA. If the test is positive, the entire genomic DNA, not sheared or cut, is pulled down. After the nickel-bead step, SYBR® Green (Thermo Fisher) is added directly to the pulled-down sample. When SYBR Green binds dsDNA, it emits strong fluorescence, so it detects whether dsDNA has been pulled down [8]. If a positive SYBR Green signal is seen, then the target site of interest is known to be present in the sample. When this method is used to detect methicillin-resistant S. aureus [8], it gives a ≥10-fold higher fluorescent signal than does methicillin-sensitive S. aureus, demonstrating high specificity. This rapid (~30-minute) technique can potentially be used to detect almost any DNA sequence. The limit of detection is 31 ng/mL or 10 colony-forming units (CFU)/mL. As long as the gRNA sequence is specific for the pathogen of interest, this is a highly specific approach to detection.
Cas12, Cas13, and Cas14 diagnostic methods
There are numerous Cas12, Cas13, and Cas14 enzymes. Diagnostic methods have been developed using many of these enzymes, including Cas12a, Cas12b, Cas13a, Cas13b, Cas13d, and Cas14a from a wide variety of bacterial species. Some of these diagnostic techniques use several of these enzymes simultaneously, allowing multiplexing assays for the purpose of distinguishing pathogens.
Collateral cleavage
The Cas12-, Cas13-, and Cas14-based diagnostic techniques depend on a process known as collateral cleavage (also called “trans cleavage”). That is, when 1 of these enzymes binds its target as directed by gRNA, the enzyme cleaves not only the target but also other nucleic acids nearby in the solution. Indeed, during collateral cleavage, Cas12 and Cas14 enzymes cleave nearby ssDNA molecules [9-11], and Cas13 enzymes cleave nearby RNA molecules [12]. Because many molecules of a nucleic acid are cleaved in 1 reaction, these enzymatic actions lend themselves well to signal amplification schemes.
Isothermal amplification
The SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) technique has been developed for detection of RNA sequences, and DETECTR (DNA endonuclease-targeted CRISPR trans reporter) for DNA detection [10]. Both SHERLOCK and DETECTR require isothermal amplification of the target sequence before the CRISPR cleavage step to obtain sufficient quantities of relevant sequences for detection. In SHERLOCK, the isothermal amplification step is followed by T7 transcription to generate RNA. In DETECTR, T7 transcription is not performed, as DNA is targeted instead of RNA. Standard DETECTR uses LbCas12a, which requires a PAM site, whereas SHERLOCK uses 1 or more Cas13 enzymes and other enzymes, removing the requirement for a PAM site [13]. There is also a modification of DETECTR that uses Cas14a, which too removes the need for a PAM site [9].
Both the DETECTR and the SHERLOCK systems use nucleic acid probes (DNA probes for DETECTR, RNA probes for SHERLOCK) with detector systems. For example, the probes may have biotin at 1 end and FAM fluorophore at the other end. The nucleic acid probes are cleaved by the Cas enzymes, allowing separation of the 2 ends. For the case of FAM- and biotin-labeled probes, results can be visualized on a lateral flow strip, where a positive signal shows 2 bands; a negative signal has only 1 band. Other probe detector systems, such as those that use various fluorophores, may be read in other ways, for example, by using fluorescence plate readers.
DETECTR has been shown to differentiate between 2 forms of human papilloma virus (HPV) [10]. SHERLOCK has been shown to detect Zika and dengue viruses, and to detect different mutations in liquid biopsies from non-small cell lung cancer patients [13].
For large-scale multiplexed virus detection, the Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN) system has been developed and shows much promise [14]. Using detection by a Cas13-based method, CARMEN allows 1 array to test over 4500 CRISPR targets.
Future promise of CRISPR for disease diagnosis
To date, publications on CRISPR-based diagnostic methods have mostly been limited to the detection of infectious diseases. However, publications on CRISPR diagnostic methods for inherited and other non-infectious diseases and cancers are starting to appear; some methods use innovative variations of CRISPR screening approaches [15-18]. CRISPR screening, which previously has been used primarily to determine possible mutation sites that would afterward need to be validated, has recently been investigated as a novel approach to determining an individual patient’s specific mutations in tumors [19]. In the rapidly advancing field of CRISPR research, applications to personalized diagnostics are likely in the near future.
