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Jolany vangah et al. Biological Procedures Online (2020) 22:22 https://doi.org/10.1186/s12575-020-00135-3 REVIEW Open Access CRISPR-Based Diagnosis of Infectious and Noninfectious Diseases Somayeh Jolany vangah1†, Camellia Katalani2†, Hannah A. Boone3†, Abbas Hajizade4, Adna Sijercic3 and Gholamreza Ahmadian1* Abstract Interest in CRISPR technology, an instrumental component of prokaryotic adaptive immunity which enables prokaryotes to detect any foreign DNA and then destroy it, has gained popularity among members of the scientific community. This is due to CRISPR’s remarkable gene editing and cleaving abilities. While the application of CRISPR in human genome editing and diagnosis needs to be researched more fully, and any potential side effects or ambiguities resolved, CRISPR has already shown its capacity in an astonishing variety of applications related to genome editing and genetic engineering. One of its most currently relevant applications is in diagnosis of infectious and non-infectious diseases. Since its initial discovery, 6 types and 22 subtypes of CRISPR systems have been discovered and explored. Diagnostic CRISPR systems are most often derived from types II, V, and VI. Different types of CRISPR-Cas systems which have been identified in different microorganisms can target DNA (e.g. Cas9 and Cas12 enzymes) or RNA (e.g. Cas13 enzyme). Viral, bacterial, and non-infectious diseases such as cancer can all be diagnosed using the cleavage activity of CRISPR enzymes from the aforementioned types. Diagnostic tests using Cas12 and Cas13 enzymes have already been developed for detection of the emerging SARS-CoV-2 virus. Additionally, CRISPR diagnostic tests can be performed using simple reagents and paper-based lateral flow assays, which can potentially reduce laboratory and patient costs significantly. In this review, the classification of CRISPR- Cas systems as well as the basis of the CRISPR/Cas mechanisms of action will be presented. The application of these systems in medical diagnostics with emphasis on the diagnosis of COVID-19 will be discussed. Keywords: CRISPR-Cas , COVID-19, Diagnostic test, SHERLOCK, DETECTR, Single guide RNA (sgRNA) Introduction deeper understanding of the structure as well as the Since the discovery of the clustered regularly interspaced function of CRISPR and CRISPR-associated (Cas) pro- short palindromic repeats (CRISPR) locus in Escherichia teins. CRISPR-Cas systems are found in prokaryotic coli (E. coli) in 1987, [35] CRISPR has revolutionized cells, both bacteria and archaea, where their main role is both research and practical achievements in biology, the protection of the organism against the introduction particularly in the areas of genome editing and genetic of exogenous DNA, such as plasmids and bacterio- engineering. CRISPR-associated genes (cas genes) were phages. These systems provide adaptive immunity to identified in 2002 [37] and further research has led to a prokaryotes, which specifically destroy the foreign DNA molecules [6]. In the last few years, different CRISPR- * Correspondence: [email protected] Cas systems have been identified or engineered, each † Somayeh Jolany vangah, Camellia Katalani and Hannah A. Boone containing distinct characteristics and applications. contributed equally to this work. 1Department of Industrial and Environmental Biotechnology, National These studies have generated many CRISPR toolboxes Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran P.O.BOX: and many CRISPR-based technologies have emerged. 14155-6343, Iran The generation of in vivo and in vitro models [31, 63, Full list of author information is available at the end of the article © The Author(s). 2020, corrected publication 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/ licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Jolany vangah et al. Biological Procedures Online (2020) 22:22 Page 2 of 14 65] developing molecular tools for genome manipulation CRISPR-Cas Biology and Characteristics [48, 81] and antiviral and antibacterial drug development CRISPR loci are composed of repeat sequences, with [1] are some of the main technologies based on CRISPR- alengthofabout20–40 bp, which are separated by Cas systems. unique interspaced 20–58 bp sequences called spacers The main breakthrough within the field of genomic [34]. A series of sequences are located downstream of engineering was the demonstration of CRISPR-Cas the CRISPR locus which code for Cas proteins. At in vitro genome editing [39]. After that, this tech- the time of their discovery, it was shown that some nique was successfully exploited by different research of these proteins were those previously thought to be groups for the manipulation of many genomes, in- involved in DNA repair [6]. Several different classes cluding mice [86], Drosophila [8], Caenorhabditis ele- of Cas proteins with a variety of activities, including gans [21], humans [15, 17, 39, 69], zebrafish [32], nuclease, helicase and polymerase activities which can bacteria [38], rice [88], etc. be exploited for nucleic acid manipulation have been Because of the need for precise diagnosis in many dis- described. The CRISPR-Cas system is responsible for ease situations, another important application of the the adaptive immune response of prokaryotes [50]. CRISPR-Cas system has emerged: detection of diseases Following the entry of viral DNA or plasmids into and microbes [33, 49]. Although robust and potent plat- the cell for the first time, it will be degraded and forms were developed based on CRISPR-Cas9 [62, 90], inserted into specific sites between repeated sequences the discovery of Cas13a (formerly C2c2) [70] and Cas12a byCasproteinsandtheCRISPRarraywillbeorga- (formerly Cpf1) [89] which both have collateral cleavage nized (Fig. 2a). activity has revolutionized the field of nucleic acid detec- However if the bacterial host does have a CRISPR sys- tion. Cas13a is a single-component, RNA-guided and tem, these sequences are transcribed in the cell to gener- targeting enzyme, which is specific for ssRNA and collat- ate CRISPR RNA (crRNA). Subsequently the crRNA erally cleaves neighbor non-targeted RNAs. In contrast, accompanying Cas proteins forms an interfering com- Cas12a is an RNA-guided, DNA-targeting enzyme which plex. The complex, under crRNA guidance, directs for- targets DNA and collaterally cleaves ssDNA (45). Differ- mation of hydrogen bonding between the crRNA and ent platforms have been developed based on these two the viral DNA that catalyzes the breaking apart of for- proteins. Specific high sensitivity enzymatic reporter eign DNA (Fig. 2b). Therefore, the infection essentially unlocking (SHERLOCK) was introduced by Gootenberg has ended before it starts. In fact, the CRISPR-Cas sys- in 2017 [25] which exploits Cas13a for the detection of tem acts like a bacterial immune system with the spacers RNA molecules and a diagnostic platform based on this in the CRISPR array presenting a history of old infec- method was developed in 2018 [24]. At the same time, a tions [34, 44]. Cas-12a-based diagnostic tool called one-hour low-cost multipurpose highly efficient system (HOLMES) was in- troduced in 2017 [14] and a diagnosis platform was de- Classification of CRISPR-Cas Systems scribed in 2018 [45]. A striking example of the power of Co-evaluation of specified anti-CRISPR proteins these systems is the extremely fast development of a encoded by invading viruses furthermore progress the CRISPR-Cas-based diagnostic test which can rapidly and ability of innate immunity in archaeal and bacterial with a high sensitivity diagnose SARS-CoV-2, an emer- organisms, leading to remarkable diversity and fast ging virus responsible for the COVID-19 pneumonia dis- adaptive evolution in Cas systems [44, 76]. Classifica- ease [11, 19, 49]. This demonstrated the high potential tion ofCRISPR-Cas systems was just emphasized on of CRISPR-Cas systems for the development of rapid de- evolutionary relationships. Therefore combination of tection of newly emerging diseases. Figure 1 shows the several criterion have recruited for classification main milestones in the research and development of schemes that are similar between conserved Cas pro- CRISPR-Cas systems with a focus on the role of these teins using: clustering and phylogenetic algorithm, systems in medical diagnosis. architectural organization in the effector modules, the The biology of the CRISPR-Cas systems, their charac- phylogeny of Cas1 and the presence of exclusive sig- teristics, and classification will be summarized in this re- nature gene in the CRISPR-Cas

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