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CRISPR-Based Strategies in Infectious Disease Diagnosis and Therapy

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CRISPR-Based Strategies in Infectious Disease Diagnosis and Therapy Infection https://doi.org/10.1007/s15010-020-01554-w REVIEW CRISPR‑based strategies in infectious disease diagnosis and therapy Alexandra Binnie1,2,3,4 · Emanuel Fernandes2,3 · Helder Almeida‑Lousada2,3,4 · Ramon Andrade de Mello2,3,5 · Pedro Castelo‑Branco2,3,4,6 Received: 19 June 2020 / Accepted: 6 November 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2021 Abstract Purpose CRISPR gene-editing technology has the potential to transform the diagnosis and treatment of infectious diseases, but most clinicians are unaware of its broad applicability. Derived from an ancient microbial defence system, these so-called “molecular scissors” enable precise gene editing with a low error rate. However, CRISPR systems can also be targeted against pathogenic DNA or RNA sequences. This potential is being combined with innovative delivery systems to develop new therapeutic approaches to infectious diseases. Methods We searched Pubmed and Google Scholar for CRISPR-based strategies in the diagnosis and treatment of infectious diseases. Reference lists were reviewed and synthesized for narrative review. Results CRISPR-based strategies represent a novel approach to many challenging infectious diseases. CRISPR technolo- gies can be harnessed to create rapid, low-cost diagnostic systems, as well as to identify drug-resistance genes. Therapeutic strategies, such as CRISPR systems that cleave integrated viral genomes or that target resistant bacteria, are in development. CRISPR-based therapies for emerging viruses, such as SARS-CoV-2, have also been proposed. Finally, CRISPR systems can be used to reprogram human B cells to produce neutralizing antibodies. The risks of CRISPR-based therapies include of-target and on-target modifcations. Strategies to control these risks are being developed and a phase 1 clinical trials of CRISPR-based therapies for cancer and monogenic diseases are already underway. Conclusions CRISPR systems have broad applicability in the feld of infectious diseases and may ofer solutions to many of the most challenging human infections. Keywords CRISPR gene editing · Infectious diseases · Viral infections · Resistant bacteria · Pandemic viruses Introduction * Alexandra Binnie The discovery of CRISPR [email protected] 1 Department of Critical Care, William Osler Health System, “Clustered regularly interspaced palindromic repeats” Etobicoke, ON, Canada (CRISPR) were first discovered in the archaea species, 2 Faculty of Medicine and Biomedical Sciences, University Haloferax mediterranei, by a Spanish microbiologist, Dr. of Algarve, Edifcio 2, Ala Norte, Campus Gambelas, Francisco Mojica, They comprised palindromic sequences 8005-139 Faro, Portugal of 30 base pairs (bp) interspersed with spacer sequences of 3 Algarve Biomedical Center Research Institute, Faro, Portugal 36 bp [1]. Initially the discovery did not generate interest 4 Centre for Biomedical Research, University of Algarve, Faro, outside the feld of microbiology, however, when similar Portugal palindromic repeats were identifed in multiple bacterial 5 ONCOLOGY PRECISION & HEALTH ECONOMICS species, including Escherichia coli, Mycobacterium tuber- RESEARCH GROUP (ONCOPRECHE), Departamento culosis, and Clostridium difcile [2], it became apparent that de Oncologia Clínica da Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, CRISPR repeats might serve an important conserved func- Brasil, & Pós-graduação em Medicina da Universidade Nove tion. Protein-coding genes, known as “CRISPR-associated de Julho(UNINOVE), São Paulo, Brasil proteins” or Cas proteins, with nucleic acid unwinding and 6 Champalimaud Research Program, Champalimaud Centre for the Unknown, Lisbon, Portugal Vol.:(0123456789)1 3 A. Binnie et al. cleavage functions were also discovered in proximity to the the pre-crRNA by a Cas protein produces the crispr-ribo- CRISPR sequences [2], but their role remained a mystery. nucleic acids (crRNAs), each of which contains a single The function of CRISPR repeats was finally eluci- spacer and a single CRISPR repeat [5]. dated in 2003, when Dr. Mojica made a key observation. To eliminate invading pathogens, the crRNA segments While searching for homologies between CRISPR spac- are released into the cytoplasm where they recognize and ers and other known sequences, Mojica identifed matches bind matching DNA or RNA sequences. When a match between some CRISPR spacer sequences and bacterio- is made, a cleavage complex is assembled containing the phages, which are viruses that infect bacteria. Mojica pos- crRNA and its target sequence, Cas nucleases, and a small tulated that CRISPR sequences and their associated Cas pro- RNA called the trans-activating CRISPR RNA (tracr- teins might function as a microbial defense system, storing RNA). This complex cleaves the target at a site adjacent to “memories” of bacteriophages, which could then be used to the crRNA target sequence, called the protospacer adjacent fght subsequent infections [1, 3]. motif (PAM) (Fig. 1). The mechanism of CRISPR function in bacteria is Cas proteins from diferent bacterial species mediate remarkably simple (Fig. 1). When a bacterium is invaded nucleic acid cleavage in diferent ways. Some Cas proteins, by a bacteriophage or plasmid, portions of the invading like Cas9, work individually, while others form cleavage DNA are cleaved by Cas proteins and inserted into the complexes containing multiple Cas proteins. Irrespec- CRISPR locus, creating a “memory” of the sequence [4]. tive of the mechanism of cleavage however, the CRISPR The bacterium constitutively transcribes the CRISPR system acts like an microbial defense system, targeting locus to produce an RNA message that contains all of the the DNA or RNA of invading agents for degradation and CRISPR repeats and their spacers. This transcript is called elimination. the pre-crispr-ribonucleic acid (pre-crRNA). Cleavage of Fig. 1 CRISPR/Cas9 function in bacteria. a Genetic material from a virus, phage, or plasmid enters the bacterium. b Short segments of genetic informa- tion from the invading agent are inserted into the CRISPR region interleaved with spacer segments. c crRNA segments consisting of CRISPR repeats and spacers are constitutively expressed in the bacterium. d Invading nucleic acid segments are recognized by crRNAs, leading to assembly of a cleav- age complex containing the foreign DNA, crRNA, and Cas9 protein and resulting in cleavage of the invading DNA 1 3 CRISPR-based strategies in infectious disease diagnosis and therapy CRISPR and gene editing a homology repair template into the cells along with the CRISPR system, the cleaved DNA ends could be repaired In 2013, Dr. Feng Zhang at the Massachusetts Institute of via homology-directed repair (HDR), in a template-specifc Technology published the frst report of CRISPR-based manner. Using this strategy, Zhang and his team were able to human gene editing [6]. His work was based on the ground- induce cleavage and repair via HDR at the EMX1 homeobox breaking studies of Emmanuelle Charpentier and Jen- gene in human cells [6]. nifer Doudna, who had previously identifed the key ele- The principle advantages of CRISPR gene editing, ments required for CRISPR-mediated cleavage [7]. Zhang relative to other gene-editing techniques, are its specifc- and his team recognized the potential of CRISPR systems to ity and accuracy of editing, even for very small sequences. produce highly targeted modifcations in human genes and This is a product of the strong complementarity between designed a method to express guide RNAs (the equivalent the guide sequences (crRNAs) and their targets, as well as of the crRNAs), tracrRNA, and the Cas9 cleavage protein the precision of Cas cleavage at the target site [8, 9]. In in human cells [6]. addition, CRISPR has the advantage of simplicity, in that One of the key issues resolved by Zhang and his team it requires, at a minimum, only a crRNA guide sequence, was how to repair the cleaved DNA at the target site. In the the Cas9 nuclease protein (from Streptococcus pyogenes), absence of intervention, repair of the cleaved DNA ends and a tracrRNA, to be functional [9, 10]. Additional func- occurs via non-homologous end joining (NHEJ) (Fig. 2). tionality is available through the use of CRISPR-associated This is an error-prone process, often resulting in insertion nucleases from diferent microorganisms, such as the Cas13 or deletion of a small number of nucleotides (indels), and protein (from Leptotrichia shahii), which is an RNA-spe- leading to unpredictable changes in the gene sequence. As cifc nuclease [8]. Cas proteins have also been engineered to a result, the target gene may or may not be inactivated after perform diferent enzymatic functions, such as DNA meth- repair. Zhang and his team postulated that by introducing ylation and base editing. These modifcations have greatly Fig. 2 Mechanisms of CRISPR/ Cas9 gene editing. a A con- struct that expresses a crRNA segment and the Cas9 protein is introduced into the target cell. b The target DNA is bound by the crRNA and Cas9 complex. c A double-strand break is introduced at the target site by the Cas9 nuclease. d The free ends of the DNA are subse- quently repaired by the cell’s DNA repair mechanisms, either through error-prone non-homol- ogous end joining (NHEJ) or, with the addition of a homology template, through homology- directed repair (HDR) 1 3 A. Binnie et al. expanded the nucleic-acid modifying potential of CRISPR- and Dengue [15, 20]. Recently, detection of SARS-CoV-2 based systems
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