Gene Editing with CRISPR-Cas9 PDF

8.6 Expanding the Possibilities of Gene Editing with CRISPR-Cas 157 Fig. 8.3 Possible delivery strategies for the CRISPR-­ Cas9 system.The different components of the CRISPR-­ Cas9 system can be provided in multiple forms. Cas9 can be delivered as a Cas9 cDNA-containing plasmid, as an mRNA molecul...

8.6 Expanding the Possibilities of Gene Editing with CRISPR-Cas 157 Fig. 8.3 Possible delivery strategies for the CRISPR-­ Cas9 system.The different components of the CRISPR-­ Cas9 system can be provided in multiple forms. Cas9 can be delivered as a Cas9 cDNA-containing plasmid, as an mRNA molecule or as a recombinant protein, already complexed with the guide RNA molecule(s). Apart from this route, the guide RNA can also be delivered in an unconjugated form to cells, as well as in the form of DNA plasmids. dCas9 fused to a transcriptional activator such as VP64 can be directed to a particular gene, recruiting transcriptional machinery that will lead to an increase of the expression levels of that gene. Conversely, dCas9 conjugated with a transcriptional repressor such as the Krüppel-­ associated box (KRAB) domain can be used to decrease the expression of a target gene, without introducing a DNA DSB and thus preventing the possibility of an undesirable indel mutation. Transcription regulation can also be achieved using dCas9 fused to epigenetic modifiers that alter the acetylation levels of histones, or the methylation levels of the DNA, or pairs of dCas9 fused with proteins that are able to interact and thereby “bend” the chromatin, generating loops in its topology [32]. Fusing fluorescent proteins such as the green fluorescent protein (GFP) can be used to pinpoint the localization of a particular genetic sequence in a chromosome. What is more, mutation of the target genome has also been shown to be achievable without recurring to DNA DSBs. Fusing deaminase proteins such as APOBEC1 along with excision repair inhibitor UGI to dCas9 or nCas9 has been shown to produce direct conversion between nucleotides. This type of strategy, termed base 158 8 Gene Editing Fig. 8.4 Versatility of the CRISPR-Cas9 system. The figure highlights possible applications of the CRISPR-­ Cas9 system that go beyond the introduction of DNA double-strand breaks. These application are based on the use of partially (nCas9) or completely (dCas9) catalytically inactivated Cas9 and include transcription activation or repression, epigenetic modulation and base editing, among others. editing, has been successfully employed in ­converting cytosines to thymines and adenines to guanines [36, 37]. In addition to this search to expand what Cas9 can do, scientists have also looked for ways to reliably control when and where its action takes place. In fact, several variants of inducible Cas9 have also been developed. Among them, some respond to chemical compounds and others to ligand-binding, and still others are activated by optical light [32]. In recent years, reengineering of Cas9 and even of the gRNA sequences has yielded a vast array of tools that is continuously being expanded 8.7 Limitations and Challenges to Current Gene Editing Strategies 159 on and that is thus optimizing the applications of the CRISPR-Cas9 system. high versatility are important factors in this ongoing search for the “ultimate” Cas protein. 8.6.2 8.7 Cas Variants The Cas9 protein from S. pyogenes is not the only Cas that has drawn the attention of scientists looking at improving the existing gene editing platforms. Many more Cas proteins have been identified and more or less extensively characterized, leading to the creation of an ever-expanding Cas protein library. Its remaining members, although not having such a widespread use as SpCas9, certainly contribute to the ever-growing possibilities offered by the CRISPR-Cas system. Different Cas proteins, including different Cas9 proteins from species other than S. pyogenes, have been described to be diversely prone to off-target effects and are known to display distinctive PAM requirements [32]. Some of these PAMs are longer than three nucleotides, and although this restricts the range of targets the respective Cas proteins can have, the increase can be beneficial in further decreasing the number of off-targets. Different PAMs may also allow targeting regions that the NGG PAM requirement of SpCas9 does not allow. Different Cas proteins also display different molecular sizes, and smaller Cas variants may be advantageous regarding their delivery to cells, for example. SpCas9 is too large for its codifying DNA to be included in AAV particles along with the gRNA; as such, currently each agent has to be provided in a different AAV vector. Smaller Cas variants may be more amenable to AVV-mediated delivery. Finally, some Cas proteins have been described to target nucleic acids differently from SpCas9 [38]. Cas12a, for example, has been shown to produce staggered DNA breaks, leading to the generation of sticky ends at the cut site. Cas13a has been shown to target RNA. New Cas variants are continuously coming to light, in a constant search for a particular Cas protein, or set of Cas proteins, that may display advantages over all others. A smaller molecular size, a low incidence of off-target effects, and a Limitations and Challenges to Current Gene Editing Strategies As promising as it may be, implementation of gene editing in an experimental setting, let alone in the development of a gene therapy approach, is subject to several limitations and challenges that must be accounted for. Some of them can be avoided or controlled for, but others will require continuous investigation and development before they can be tackled with unquestionable success. Overall, challenges to the application of gene editing include (a) the ability to properly deliver the molecular tools, (b) problems with specificity, (c) guarantees of fidelity, and (d) control over DSB repair and HDR [4–6]. As explained in Chap. 4, delivery of gene therapy agents faces several physiological barriers that limit their efficacy. What is more, it is important to ensure that the molecular tools reach the proper organ or tissue where their therapeutic effect will take place. All the while, the delivery mechanisms should pose no threat to the health and safety of the organism. Selection and design of methods for the delivery of gene editing tools should abide by the same principles as other gene therapy approaches, possibly preferring ex vivo strategies over local or systemic administration as a means to improve safety and minimize delivery to unintended tissues or cells [31]. As mentioned above, none of the gene editing nuclease platforms is devoid of possible off-­ target effects, by virtue of the permissiveness every system has, on a higher or lesser degree, to mismatch tolerance. Ideally, the nuclease systems would have no off-targets, but since it is currently impossible to ensure this, it is crucial that gene editing strategies minimize the occurrence of unintended modifications. This can be done by selecting variants with a proven lower degree of off-target activity or, in the case of CRISPR-Cas, selecting gRNA sequences with low levels of predicted off-targets. After editing, it is important 8 160 to be able to search the targeted genome for putative off-target mutations. This can be done by whole-genome sequencing and through other, recently developed, targeted approaches, but such techniques may not be available to every lab. Fidelity, in the context of gene editing, describes the degree to which the intended alteration was inserted in the genome and chiefly concerns mutations introduced by HDR [6]. As a result of a DSB and in the presence of an HDR repair template, the intended insert may be introduced at a site other than the one expected, more than one copy of the insert may be introduced, and translocations may occur, among many other unintended changes with disastrous effects. Techniques that allow confirmation that only the intended changes took place are necessary. Finally, the infrequent occurrence of HDR is a limiting factor for strategies that rely on this pathway of DSB repair for the intended genome alteration to occur. HDR is limited to dividing cells, and its rate is generally low, compared to NHEJ [9]. Several lines of research have invested in increasing the rate of HDR, but, as with any other manipulation that may interfere with DNA repair mechanisms, the possibility of unexpected outcomes may outbalance the benefits of the intervention. Nonetheless, in the case of the CRISPR-Cas system, diverse approaches have been described to increase success of HDR-­ mediated editing, including employing nickases instead of wild-type Cas9, since a DNA nick is less prone to induce NHEJ-derived indels while still potentiating HDR; enriching cell cultures with cells in the G2/M phase of the cell cycle; inducing “cold shocks”; overexpressing the Rad51 protein; employing small molecules and other chemical compounds (RS-1, Brefeldin A, L755507, Nocodazole); and fusing Cas9 with proteins involved in HDR, among many others [39–43]. Studies aiming to define the best parameters for donor repair template design are also ongoing. 8.8 Gene Editing  ene Editing as a Tool G for Human Disease Therapy The possibilities offered by existing gene editing strategies can be translated into diverse approaches to tackle human health conditions and disease. Importantly, gene editing may be a significant tool not only for direct therapeutic intervention but also in other, no less relevant, steps of the therapy development pipeline [44]. Nuclease-based gene editing can be employed in basic research, assisting in the investigation of gene functions. The CRISPR-Cas system, in particular, can be used to perform high-throughput screening of disease modifiers, which may yield important clues into disease pathogenesis and possibly constitute relevant therapeutic targets [45]. Gene editing is also a potent method for generating disease models and can be used to develop isogenic cell lines, i.e., cell cultures derived from individuals, where disease-causing genes can be introduced or removed, thus producing cultures with the same genetic background, in which the only modifying factor is the designated genetic factor [46]. Additionally, gene editing allows for the rapid and inexpensive generation of animal models, compared with traditional methods used for generating transgenic knockout, and knock-in animals [35]. CRISPR-­ Cas9 is particularly well-suited for modeling complexed diseases, by allowing the alteration of several genes simultaneously through its multiplexing capability [47]. Concerning the use of gene editing as a therapeutic approach, the interest this field is drawing has led research teams to develop and test diverse gene therapy strategies that are based on the growing capabilities of the described systems to operate changes in the DNA. Current literature offers diverse examples, overall focusing on one of several routes: (a) correcting pathogenic mutations; (b) inactivating diseasecausing genes; (c) re-establishing gene functions; (d) eliminating disease-causing elements; (e) introducing protective mutations; and (f) Review Questions generating cells with therapeutic activities. Examples of the potential of these approaches abound in reports using both cell cultures and animal models, and some strategies have already transitioned to clinical testing. Two examples of gene editing-based approaches to therapy follow: one focusing on a genetic disorder and another on an infectious disease. As described in Chap. 7, Duchenne muscular atrophy (DMD) is a hereditary disorder that arises as a consequence of mutations in the DMD gene, which in healthy individuals codifies dystrophin, a protein that plays a crucial role in muscular structure and physiology (Fig. 7.4). Amoasii and collaborators systemically administered AVVs codifying CRISPR-Cas tools to dogs harboring a deletion of exon 50 of the DMD gene, which is a hot spot for mutations linked to DMD, in humans. CRISPR-Cas activity was directed at an early region of exon 51. Overall, treated animals displayed an improvement in muscular histology and dystrophin levels. Indels were detected at the targeted genomic site, and the amelioration observed was related to reestablishment of the reading frame of the gene or exon 51 skipping [48]. Human immunodeficiency virus (HIV) infection of T-cells relies on interaction of the virus with cell receptors that mediate its internalization (Fig. 3.4). As explained in Chap. 3, one of these receptors is CCR5, and it has been known for some time that individuals bearing mutated forms of this receptor are refractory to HIV infection. It has been thus hypothesized that knocking out CCR5 gene function through the action of programmable nucleases may be beneficial in preventing HIV entry into lymphocytes [49, 50]. In 2014, in the very first clinical trial using gene editing, HIV patient cells were edited ex vivo with ZFNs and then autologously reinfused, with promising results in what regards to viral loads in circulation [51–53]. Other clinical trials using similar rationales were also underway at the time, and others followed, with a clinical trial for HIV using CRISPR-Cas-edited stem cells being currently underway [54]. 161 This Chapter in a Nutshell • Current gene editing strategies rely on two conditions, (i) the ability to define the specific region of the genome to be altered and (ii) the ability to create conditions for the desired alterations to occur. • The definition of the target site is accomplished by molecules that specifically bind to a nucleotide sequence and then cleave the DNA producing a double-strand break (DSB). These are then repaired through different endogenous mechanisms of DNA repair. • There are two main mechanisms of DNA DSB repair, nonhomologous end-joining (NHEJ) and homology-direcgted repair (HDR), with the former being simpler and the latter involving a DNA molecule as template for the repair. • Four main systems of programmable nucleases have been used in gene editing: meganucleases, zinc-finger nucleases, TALENs, and the CRISPR-Cas system. • The CRISPR-Cas system presents advantageous features over the other programmable nuclease systems. Several variations of the system have been developed, or example in order to alter epigenetic markers, the architecture of the chromatin and the levels of transcribed RNA molecules. • Despite being an important promise for gene therapy, gene editing faces important limitations and challenges. Some are also found in other gene therapy strategies, but other are especific to this approach. Review Questions 1. In the context of gene therapy, gene editing systems can be used to: (a) Disrupt a mutated gene (b) Substitute a malfunctioning gene (c) Regulate the expression of a mutated gene (d) None of the above (e) All of the above