G C A T T A C G G C A T genes Review Therapeutic Editing of the TP53 Gene: Is CRISPR/Cas9 an Option? Regina Mirgayazova 1, Raniya Khadiullina 1, Vitaly Chasov 1, Rimma Mingaleeva 1, Regina Miftakhova 1, Albert Rizvanov 1 and Emil Bulatov 1,2,* 1 Kazan Federal University, 420008 Kazan, Russia; [email protected] (R.M.); [email protected] (R.K.); [email protected] (V.C.); [email protected] (R.M.); [email protected] (R.M.); [email protected] (A.R.) 2 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia * Correspondence: [email protected] Received: 6 May 2020; Accepted: 23 June 2020; Published: 25 June 2020 Abstract: The TP53 gene encodes the transcription factor and oncosuppressor p53 protein that regulates a multitude of intracellular metabolic pathways involved in DNA damage repair, cell cycle arrest, apoptosis, and senescence. In many cases, alterations (e.g., mutations of the TP53 gene) negatively affect these pathways resulting in tumor development. Recent advances in genome manipulation technologies, CRISPR/Cas9, in particular, brought us closer to therapeutic gene editing for the treatment of cancer and hereditary diseases. Genome-editing therapies for blood disorders, blindness, and cancer are currently being evaluated in clinical trials. Eventually CRISPR/Cas9 technology is expected to target TP53 as the most mutated gene in all types of cancers. A majority of TP53 mutations are missense which brings immense opportunities for the CRISPR/Cas9 system that has been successfully used for correcting single nucleotides in various models, both in vitro and in vivo. In this review, we highlight the recent clinical applications of CRISPR/Cas9 technology for therapeutic genome editing and discuss its perspectives for editing TP53 and regulating transcription of p53 pathway genes. Keywords: TP53; mutation; CRISPR/Cas9 gene editing; base editing; prime editing; epigenome regulation; clinical trial 1. Introduction The TP53 gene encodes the p53 protein, a well-known tumour suppressor involved in various regulatory pathways including cell cycle arrest, apoptosis, senescence and DNA repair. The TP53 is the most frequently mutated gene in human cancer with mutations found in about half of all cancer cases. The Genomics Evidence Neoplasia Information Exchange (GENIE) project by the American Association for Cancer Research (AACR) reports TP53 mutations in 32.8% of cancers, primarily lung adenocarcinoma, breast invasive ductal carcinoma and colon adenocarcinoma (Figure1)[1]. Genes 2020, 11, 704; doi:10.3390/genes11060704 www.mdpi.com/journal/genes Genes 2020, 11, 704 2 of 16 Genes 2020, 11, 704 2 of 17 Genes 2020, 11, 704 2 of 17 FigureFigure 1. 1.Top Top 10 10 disease disease cases cases withwith TP53TP53 mutations, according according to to th thee GENIE GENIE (Genomics (Genomics Evidence Evidence NeoplasiaNeoplasia Information Information Exchange) Exchange) project project by by AACR AACR (American (American Association Association for for Cancer Cancer Research). Research). Figure 1. Top 10 disease cases with TP53 mutations, according to the GENIE (Genomics Evidence Currently,Currently,Neoplasia there there Information are are over over 170Exchange) 170 ongoing ongoing project clinical clinical by AAC trials Rtrials (American mostly mostly forAssociation for malignant malignant for Cancer solid solid tumours,Research). tumours, non-small non- cellsmall lung cell carcinoma, lung carcinoma, breast carcinoma, breast carcinoma, ovarian carcinomaovarian carcinoma and acute and myeloid acute leukaemia myeloid leukaemia in which TP53 in statuswhich serves TP53Currently, asstatus an inclusion thereserves are as over eligibility an inclusion170 ongoing criteria eligibility clinical [1]. Among trialscriteria mostly them [1]. Amongfor 66 aremalignant phase them I,solid66 99 are aretumours, phase phase I,non-II, 99 10 are are phasephasesmall III II, and 10cell are1 lung is phase phase carcinoma, III IV. and 1breast is phase carcinoma, IV. ovarian carcinoma and acute myeloid leukaemia in which TP53 status serves as an inclusion eligibility criteria [1]. Among them 66 are phase I, 99 are AlterationsAlterations in in the theTP53 TP53gene gene are are commonly commonly associated associated with with increased increased risk risk of cancer. of cancer. In most In most cases, phase II, 10 are phase III and 1 is phase IV. cases, only one nucleotide is mutated inTP53 the TP53 gene leading to a single amino acid substitution in only oneAlterations nucleotide in is the mutated TP53 gene in the are commonlygene leadingassociated to with a single increased amino risk acid of cancer. substitution In most in the the p53 protein. The most frequent mutations are represented by G:C>A:T or G:C>T:A conversions p53 protein.cases, only The one most nucleotide frequent is mutated mutations in the are TP53 represented gene leading by G:Cto a >singleA:T oramino G:C acid>T:A substitution conversions in that that together amount for over half of TP53 somatic mutations in human cancers (Figure 2) [2]. Often togetherthe p53 amount protein. for The over most half frequent of TP53 mutationssomatic mutations are represented in human by G:C>A:T cancers or (Figure G:C>T:A2)[ conversions2]. Often these mutationsthesethat mutations together are frameshift amountare frameshift for and over results andhalf ofresults inTP53 non-functional somaticin non-functional mutations protein inprotein human with with cancers impaired impaired (Figure transcriptional transcriptional2) [2]. Often and tumourandthese tumour suppressor mutations suppressor activity.are frameshift activity. and results in non-functional protein with impaired transcriptional and tumour suppressor activity. FigureFigure 2. Types 2. Types and and distribution distribution of of nucleotide nucleotide conversions conversions inin TP53TP53 that result result in in somatic somatic mutations mutations in Figure 2. Types and distribution of nucleotide conversions in TP53 that result in somatic mutations humanin cancers,human cancers, according according to the IARCto the (InternationalIARC (International Agency Agency for Research for Research on Cancer) on Cancer)TP53 Database.TP53 in human cancers, according to the IARC (International Agency for Research on Cancer) TP53 Database. Database. Genes 2020, 11, 704 3 of 16 In addition to the loss of the tumour suppressor functions, mutant p53 proteins may also promote the division of cancer cells. Shelley Berger with colleagues [3] have shown that this “gain-of-function” mutant p53 targets epigenetic regulators such as MLL1, MLL2 and MOZ, resulting in uncontrolled cell replication. This mainly affects late stage and hard to treat cancers such as pancreas, breast, brain, oesophagus, head and neck. Interestingly, there are many cancers with WT TP53 in which the p53 protein is inactive. Oncogenic growth of such tumours is associated with reduced p53 activity caused by overexpression of one or more epigenetic regulators that post-translationally modify p53 protein and impair its tumour-suppressor functions, including Aurora A kinase that phosphorylates p53 (S212/S312) and methyltransferases that methylate p53 such as Smyd2 (K370), Glp/G9a (K373) and PR-Set7 (K382). Inhibition of these epigenetic regulators could lead to reactivation of p53 in cancer [4]. Currently, therapeutic approaches specifically targeting the p53 protein and its negative regulators, such as MDM2, are mostly focused on small molecule modulators [5–8]; however, the emerging genome-editing technologies might soon turn into a tantamount alternative. Gene editing as a molecular technology provides the capability to change genomic sequences in almost all types of cells. Typically, this represents DNA modifications, such as insertions, deletions and sequence substitutions, at specific locations in the genome. Several primary approaches to programmable genome editing include zinc-finger nucleases (ZFNs) [9,10], transcription activator-like effector nucleases (TALENs) [11,12] and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas (CRISPR-associated) enzymes (Table1)[ 13,14]. Other examples include ZFNs and TALENs, the fusion proteins consisting of DNA-binding and nuclease domains of restriction enzyme FokI, that were among the first classes of nucleases developed for precision gene modifications. However, the application of these technologies is often limited, because they are based on proteins to guide the gene editing machinery to specific DNA sequences. Therefore, targeting each new genome site requires engineering and cloning of a new protein. Currently, the CRISPR/Cas gene-editing platform based on bacterial antiviral defence mechanisms is considered to be the most prominent and elegant RNA-guided tool for efficient and specific DNA modifications. Unlike TALENs and ZFNs, DNA recognition by the CRISPR/Cas system is specified by the short-guide RNA sequence which makes this molecular tool simple and facilitates its universal use. The ultimate objective for various genetic tools is in their clinical application, since they hold a great potential for treating human diseases, especially genetic disorders and cancer. Table 1. The classification of CRISPR/Cas systems. Interference Stage Effector Module Pre-crRNA Processing (Target Cleavage) Type I Cas3 Multi-subunit Subunit of the Class 1 Type III Cas6 effector complex processing complex Type IV Uncharacterized Type II RNase III Single multi- Nuclease domain of the Class 2 Type V domain protein