Gene Editing (PDF)

8 Gene Editing 8.1 Rewriting the Typewriter For many decades, scientists have tried to develop methods to modify the fundamental code that underlies every single organism – the genome. The reasons behind this intent have been diverse: altering genes and proteins in order to study their roles an...

8 Gene Editing 8.1 Rewriting the Typewriter For many decades, scientists have tried to develop methods to modify the fundamental code that underlies every single organism – the genome. The reasons behind this intent have been diverse: altering genes and proteins in order to study their roles and functions; modifying cells and organisms with scientific, or commercial interest; or developing strategies and approaches to tackle human diseases, among many others. Beginning in the 1920s, scientists have made use of electromagnetic radiation, mutagenic chemical compounds, recombinant DNA and molecular cloning technology, transfection methods, viruses and transposons in order to alter the nucleic acids inside a cell, generating random mutations, inserting or removing genes, or modifying existing ones [1]. Each of these techniques has revolutionized the fields of molecular biology and biotechnology and has contributed to the development of many other areas. However, the degree of precision and fidelity these techniques allow is below the ideal, perhaps utopic, scenario of being downright able to “freely rewrite” an existing genome. In eukaryotic cells, perhaps the approach that has come consistently closest to this objective relies on the exploitation of homologous recombination mechanisms as a means to target and alter specific genetic loci [2, 3]. When a DNA sequence is inserted into a cell, there is the chance © Springer Nature Switzerland AG 2020 C. Nóbrega et al., A Handbook of Gene and Cell Therapy, https://doi.org/10.1007/978-3-030-41333-0_8 that it will be randomly inserted into one of its chromosomes. However, if that DNA fragment is additionally flanked by two regions that are homologous to a particular DNA sequence of the target cell genome, it is possible that the endogenous homologous recombination mechanisms will lead to the substitution of the homologous site with the exogenous sequence, thereby inserting or substituting the region in between the homology arms. This gene targeting strategy has been recurrently and reliably applied to the generation of knockout mouse models, as well as knock-in animals in which particular genetic sequences have been altered or inserted. These types of models have come to be some of the most valuable tools in biomedical research, providing the bases for studies that have unveiled crucial aspects of human pathologies and contributing to the development and preclinical testing of therapeutic approaches. Nonetheless, this proven method of modifying genomes may not be practical, feasible, or at all possible in every type of setting. The dominion scientists have acquired over mouse genetics is not always easily translated to other organisms, which may have their own particularities regarding life cycle, reproductive behavior, development, or the very molecular mechanisms that take place inside their cells. Moreover, generation of knock-in and knockout animals relies on a series of procedures, using, for example, stem cell cultures and animal crossings, which are not 147 8 148 conceivable in several other contexts, such as when aiming at more straightforward forms of cell manipulation. Genome manipulation as a possible pathway to the treatment of human diseases should ideally target a patient’s cells directly and allow a high degree of control over the changes that are introduced. It is tempting to conjecture that techniques may be developed that, by allowing the direct rewriting of the four-letter blueprints of the human organism, will be able to delete disease-­ associated genes and correct pathogenic mutations. Over the last few years, the promise of more direct, precise, and versatile methods for “rewriting” the eukaryotic genome has increasingly drawn the attention of the scientific community. Researchers are now employing a variety of new ways to manipulate genes, collectively termed as gene, or genome, editing. These techniques offer the promise of being able to target precise regions of the genome of any cell and produce a variety of customizable modifications with an unprecedented degree of consistency. 8.2 The Basis of Gene Editing Present-day approaches to gene editing rely on two conditions: (a) the ability to define the region of the genome that is to be altered and (b) the capacity to effect the actual changes or, more precisely, to create the conditions for the desired alterations to occur [4, 5]. These two abilities combine to generate the desired modification(s), at the intended locus (or loci). Definition of the target site is accomplished by molecules that specifically bind to a particular nucleotide sequence and subsequently cleave both chains of the DNA, producing a double-­ strand break (DBS) [6]. These molecules are endonucleases – enzymes that are able to separate nucleotides adjacently localized in the middle of a polynucleotide chain, by cutting the phosphodiester bond existing between them. In order to target a particular sequence with as much specificity as possible, endonucleases used in gene editing must be as selective as possible in Gene Editing regard to the DNA sequence that they bind to and cut. The region to be altered can coincide with the sequence targeted by the endonucleases or, alternatively, that region can be in the close vicinity of the targeted sequece. Because of the central role endonucleases have in current approaches to gene editing, the term gene editing could, and perhaps should, be more appropriately substituted by “nuclease-­ based gene editing” [6]. However, DNA DSBs in isolation would be insufficient to edit genes and genomes. The actual modifications that then take place in the nucleotide sequence are in fact enacted by the cell, namely, through its endogenous DNA repair mechanisms [4, 5]. As a direct consequence of a DSB, DNA repair systems are recruited to the vicinity of the DSB site [7]. Changes to the nucleotide sequence may be introduced upon DNA repair, and, if appropriate conditions are established, those changes will result in the desired modification of the target locus. The following section briefly outlines the two main mechanisms through which DNA DSBs are repaired in a cell and the ways they can be exploited in order to generate a particular desired alteration in the genome. The section after that will describe the four main classes of endonucleases that can be used to introduce the DSBs responsible for triggering modifications at the intended sites. 8.3  NA Double-Strand Break D Repair Mechanisms Maintaining the integrity of the genome is of crucial importance for the preservation of cellular homeostasis and for the overall health of the organisms the cells compose. For this reason, Life has evolved diverse systems and molecular pathways that ensure that the DNA is appropriately repaired in case an insult threatens its integrity. DNA DSBs in particular are mainly repaired through one of two mechanisms: nonhomologous end-joining (NHEJ) and homology-directed repair (HDR; Fig. 8.1). A vast array of proteins participates in both pathways, performing intricate biochemical and 8.3 DNA Double-Strand Break Repair Mechanisms 149 Fig. 8.1 Main mechanisms of DNA double-strand break (DSB) repair. Nonhomologous end-joining (NHEJ) involves resealing of the DSB site by simple linking of the two free ends of the DNA double strand. However, this process is prone to errors and often leads to the introduction or deletion of nucleotides. Homology-­ directed repair (HDR) is a more complex mechanism, in which the DSB is repaired using a homologous DNA molecule as template. While NHEJ may be exploited in order to generate gene knockouts or delete genetic elements, HDR may be used to introduce or substitute specific nucleotide sequences in a genome. structural operations that are beyond the scope of this chapter. Nonetheless, regarding their role in gene editing, it is important to understand that, perhaps ironically, these DNA repair mechanisms are not error-proof and can thus be manipulated in order to produce intentional changes in the genome. 8.3.1 Nonhomologous End-Joining Between the two DSB repair pathways, nonhomologous end-joining (NHEJ) is the simpler mechanism, leading to the straightforward resealing of the DSB by “regluing” the free ends left at each side of the break site [7, 8]. The proteins 8 150 mediating this pathway “mend” the “wound” that was introduced in the DNA, but, importantly, a nucleotidic “scar” may be left behind. In fact, this mechanism is somewhat error-prone, considering that NHEJ machinery may introduce or delete a small number of nucleotides as part of the process of resealing the break. As a result, the nucleotide sequence at the DSB site undergoes a small mutation, which may consist of a small insertion or a small deletion of nucleotides [4, 5]. This type of mutation is termed an indel. The number of nucleotide pairs that are added or removed from the DNA chains as part of an indel varies. If the DSB occurs at an exonic region of a gene and an indel is subsequently introduced, these small insertions or deletions of nucleotides may produce alterations in the reading frame of the mRNA molecules that will be transcribed from that gene [6]. This occurs when the indel size is not a multiple of 3. A frequent consequence of such DNA frameshifts is the appearance of premature stop codons that will halt translation and thus inhibit expression of the gene targeted by the DSB. 8.3.2 Homology-Directed Repair Gene Editing vicinity of the DSB site [4]. In a normal biological context, the repair template for HDR corresponds to the sister chromatid of the one that underwent the DSB [9]. In the context of gene editing, an exogenous DNA repair template can be provided. Repair templates can be designed so as to induce precise alterations to the genome; they must include sequences bearing complete homology to regions in the vicinity of the DSB site, but they can also include an intentionally designed, and altered, sequence. Usually, these exogenous repair templates consist of a DNA sequence including two homology arms, flanking the region that is to be inserted in the vicinity of the break site or that will substitute a portion of the genome at that vicinity. Upon DSB, the HDR machinery will repair the break using the exogenous template as a model, thereby introducing the altered sequence into the genome that is being repaired. 8.3.3  anipulating DNA Double-­ M Strand Break Repair Mechanisms to Edit Genomes NHEJ and HDR define, and limit, what kind of genome alterations can be achieved through Homology-directed repair (HDR) is a more con- nuclease-based gene editing. The action of both servative, and elaborate, mechanism, whereby mechanisms can be directed to produce different DSB repair is performed using a homologous changes that may be advantageous in the context DNA molecule as a repair template [7, 9]. The of biomedical investigation and gene therapy process involves (a) the generation of single-­ development [6]. stranded DNA (ssDNA) overhangs at the break NHEJ is an exogenous template-independent site; (b) homology-directed invasion of the DNA mechanism and relies only on the ability to pretemplate by the ssDNA; and (c) synthesis of cisely define the site at which DSBs will be introDNA primed by the invading DNA strand and duced. As explained above, simply producing a using the homologous DNA duplex as a tem- DSB in the codifying region of a gene can be sufplate. Both DNA molecules are then separated ficient to knock out that gene: the DSB can prothrough one of several different possible mecha- duce an indel mutation that will generate a nisms that may, or may not, involve DNA cross- premature stop codon. In the same way, an indel over. Whatever the case, at the end of process, can be enough to restore the reading frame of a the DNA molecule that underwent DSB and gene bearing a frameshift-inducing mutation. HDR is seamlessly repaired, in the large major- Moreover, NHEJ can also be used to “excise” a ity of cases [7]. particular genetic region. Upon producing two Though HDR is generally not as error-prone DSBs, one upstream and another downstream of as NHEJ, it may also be directed to produce a region that is to be deleted, the NHEJ pathway desirable alterations of the DNA sequence at the may reseal the DNA by uniting the end upstream 8.4 Programmable Nucleases Used in Gene Editing of the first break site and the end downstream of the second, thus excluding the intervening region. Taking advantage of HDR requires not only the ability to direct the repair machinery to the target site by inducing a DNA DSB but also the provision of a homologous repair template that will bear the particular alterations to be introduced. Broadly speaking, HDR allows for both substitutions and insertions of particular nucleotide sequences. In principle, HDR can be used to introduce a mutation of one or more nucleotides, to correct a particular mutation, or to eliminate a particular gene sequence, by providing a template in which that sequence was removed. Through HDR, particular genes can be inserted in the genome: a particular therapeutic gene may be introduced at a designated site, or a particular tag or fluorescent protein can be introduced in frame with another existing gene. It must be noted, however, that this type of outline assumes that scientists would have complete control over the DSB repair mechanisms employed by the cell. This is not the current reality. The factors that determine whether DSBs are repaired through one path or the other are still being elucidated [7, 9]. Overall, NHEJ is favored over HDR, making its applications more reliable. What is more, the absence of a repair template would completely preclude HDR in favor of NHEJ, making it more reliable still. HDR-­ dependent strategies are more challenging. Given its endogenous dependence on homologous sister chromatids, HDR occurs only in dividing cells, excluding any HDR-based strategy from use on postmitotic cells. Additionally, the principles governing exogenous repair template design are still not completely clear. Although small insertions or substitutions can be reliably enacted, introduction of longer sequences is still fraught with several experimental limitations [10]. 8.4 Programmable Nucleases Used in Gene Editing Gene editing relies on endonucleases to precisely define the region of the genome that will be altered. In order for a particular class of endonu- 151 cleases to be suitable for this end, they have to possess a series of characteristics that are not transversal to all nucleases that can be found in Nature. For these reasons, while some nucleases used in gene editing are, in fact, more or less similar to their natural cognates, others are artificial chimeric proteins, engineered from naturally occurring proteins and protein domains. Nucleases used in gene editing must be able to cut both chains of a DNA duplex and be highly specific, in regard to the nucleotidic sequences they target. If that was not the case, DSBs could be inserted in several different sites of the genome at the same time, producing unintended changes. A high specificity minimizes putative off-target effects. Overall, the longer a particular base sequence that a nuclease recognizes, the greater the specificity of the nuclease, since the probability of that sequence being repeated in the genome is lower. Additionally, nucleases used in gene editing are preferably programmable, i.e., they are amenable to being redesigned and reengineered in order to target them to different gene loci, with high specificity, according to the aims of the gene editing approach at hand. Since the 1980s, four classes of endonucleases have been selected and engineered for use in gene editing approaches (Fig. 8.2; Table 8.1). 8.4.1 Meganucleases Meganucleases, also named homing endonucleases, are naturally occurring restriction enzymes that are found in diverse organisms, including bacteria, archaea, fungi, algae and plants [11, 12]. Contrary to the restriction enzymes that are routinely employed in molecular cloning, such as EcoRI or HindIII, meganucleases recognize extended base pair sequences: from 12 to 40 base pairs, contrasting with the 6 base pairs of those, and many other, traditional restriction enzymes. This long recognition sequences are responsible for the meganuclease designation and for the high degree of target discrimination these enzymes possess. Among the meganucleases most used in genome engineering are I-SceI from