key: cord-0878072-4resbd00 authors: Jamehdor, Saleh; Naserian, Sina; Teimoori, Ali title: Enhanced high mutation rate and natural selection to produce attenuated viral vaccine with CRISPR toolkit in RNA viruses especially SARS-CoV-2 date: 2021-12-14 journal: Infect Genet Evol DOI: 10.1016/j.meegid.2021.105188 sha: a372b8486904b0c2f8aa3772ab16ffc1df40d919 doc_id: 878072 cord_uid: 4resbd00 The best and most effective way to combat pandemics is to use effective vaccines and live attenuated vaccines are among the most effective vaccines. However, one of the major problems is the length of time it takes to get the attenuated vaccines. Today, the CRISPR toolkit (Clustered Regularly Inerspaced Short Palindromic Repeats) has made it possible to make changes with high efficiency and speed. Using this toolkit to make point mutations on the RNA virus's genome in a coculture of permissive and nonpermissive cells and under controlled conditions can accelerate changes in the genome and accelerate natural selection to obtain live attenuated vaccines. With the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and becoming a pandemic crisis, the need for an effective vaccine for viruses has been considerably increased considered and several research studies have been focusing on making a vaccine to prevent the disease (Alturki et al., 2020) . Attenuated vaccines are the most effective vaccines for immune system education, stimulation, and immunity (e.g. mumps, measles, vaccinia, rubella, rotavirus, and yellow fever) ("Immunology and Vaccine-Preventable Diseases," n.d.). Natural selection is one of the best ways to make an effective attenuated vaccine (Fields et al., 2013) . For this purpose, the viruses are consecutively cultured in vitro and in vivo for years to get attenuated by various random genome mutations (Sabin and Boulger, 1973) . During long passages in a variety of experimental conditions (e.g. low temperature or different cellular and animal hosts), the driving force of natural selection tries to shift the virus populations toward the highest compatibility by fixing adaptive mutations (Dolan et al., 2018; Duffy et al., 2008) . This is indeed time-consuming in vitro and ineffective on some viruses especially DNA viruses because it needs to be passaged and purified many times to get an effective live attenuated virus vaccine and difficult to control while manipulated using in vivo conditions (Dolan et al., 2018) . Therefore, a faster and more efficient system is needed more than ever. Researchers are using a new toolkit called CRISPR to make targeted changes in the genome. An RNA-designed (single guide RNA [sgRNA]) for the target point is responsible for endonuclease protein (Cas9[CRISPR-associated protein 9]), Cas12, and Cas13 guidance. By use of the CRISPR toolkit and modified CRISPR toolkit systems, we can knock-in and knock-out genes, transcription activation, and suppression, epigenetic changes, edit the base, and image-specific nucleic acids (Adli, 2018; Jamehdor et al., 2020) . Research studies have shown that this system is effective in RNA and DNA editing (Kushawah et al., 2020; Ran et al., 2013) . Although this J o u r n a l P r e -p r o o f Journal Pre-proof toolkit has worked well in viruses' genome alterations as well, it has two major drawbacks including off-target (attach to other locations and make cuts in them) and delivery of the system to the target tissue (Xu et al., 2019; Zhang et al., 2015) Type VI (Class 2 Cas proteins) systems are the only prokaryotic CRISPR-Cas immune systems known to target RNA (and not DNA) molecules exclusively, therefore, they show specific potential for RNA detection and manipulation (Konermann et al., 2018) . Cas13 is recognized in several classes in which some have been studied (Abudayyeh et al., 2016; Kushawah et al., 2020; Smargon et al., 2017) . Scientists by use of targeted point mutations in Cas13, inactivate RNA cleavage site so, it can bind to RNA but has lost the ability to cut the RNA (called deactive Cas9 [dCas9]). Various effector protein domains have been fused to dCas13 to expand the functionality of Cas13 beyond the RNA cleavage. Fusion of ADAR (adenosine deamination of RNA) enzyme domain with Cas13 resulted in RNA editing (Figure 1 ) In this perspective we think J o u r n a l P r e -p r o o f Journal Pre-proof that rate of mutation by CRISPR toolkit library is relatively high but many of these mutations are deleterious and small remaining part of mutations can be effective and functional. However, in some viruses such as influenza and SARS-CoV-2 induced mutation can be tolerated. In addition, the mutation on RNA made by CRISPR can only lead to part of amino acid substitution. Although having an off-target effect is one of the drawbacks of this technology, it is considered as an advantage since this feature is used to create more mutations in the virus genome. In RNA viruses, a wider range of mutations can be induced with the CRISPR base editing toolkit. Live attenuated vaccines are the best vaccines with a high level of creating immunity. The main problem with making these vaccines is a long time and the high cost of producing these viruses. By creating a random mutation in the genome of viruses by the CRISPR toolkit and selecting a live attenuated virus adapted to non-permissive cells, we can get these viruses at a low cost and high production speed. In addition, this system can be used to study the evolution of viruses and predict the possibility of changes in the viruses over the coming years. This system has a high potential for application in the laboratory and it is a flexible system that can be useful for manipulating a variety of viruses. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), single guide RNA ( (1) Specific identification of RNA by the dCas13 protein with the guidance of the sgRNA that has been fused to ADAR or APOBEC. (2) Random mutation in the genome of RNA viruses by CRISPR toolkit. Ethical Approval and Consent to participate Not applicable. Availability of data and material Not applicable Funding Dr. Sina NASERIAN was supported by a governmental grant via ""l'Agence Nationale de la Recherche"" in the form of ""programme d'Investissements d"avenir"" with the grant number: ANR_15-RHUS60002. All authors conceived, wrote, and approved the final manuscript. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector The CRISPR tool kit for genome editing and beyond The 2020 Pandemic: Current SARS-CoV-2 Vaccine Development Search-and-replace genome editing without double-strand breaks or donor DNA RNA editing with CRISPR-Cas13 Evidence for hostdependent RNA editing in the transcriptome of SARS-CoV-2. Sci. 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