摘要
•The development of genome editing is framed by earlier DNA-modifying technologies.•The various aspects underlying genome editing are reviewed and integrated.•The main genome editing approaches and their collateral effects are discussed.•The efforts seeking improved genome editing end-points are highlighted. Genome editing (GE) entails the modification of specific genomic sequences in living cells for the purpose of determining, changing, or expanding their function(s). Typically, GE occurs after delivering sequence-specific designer nucleases (e.g., ZFNs, TALENs, and CRISPR/Cas9) and donor DNA constructs into target cells. These designer nucleases can generate gene knockouts or gene knock-ins when applied alone or in combination with donor DNA templates, respectively. We review progress in this field, with an emphasis on designer nuclease and donor template delivery into mammalian target cell populations. We also discuss the impact that incremental improvements to these tools are having on the specificity and fidelity attainable with state-of-the-art DNA-editing procedures. Finally, we identify areas that warrant further investigation. Genome editing (GE) entails the modification of specific genomic sequences in living cells for the purpose of determining, changing, or expanding their function(s). Typically, GE occurs after delivering sequence-specific designer nucleases (e.g., ZFNs, TALENs, and CRISPR/Cas9) and donor DNA constructs into target cells. These designer nucleases can generate gene knockouts or gene knock-ins when applied alone or in combination with donor DNA templates, respectively. We review progress in this field, with an emphasis on designer nuclease and donor template delivery into mammalian target cell populations. We also discuss the impact that incremental improvements to these tools are having on the specificity and fidelity attainable with state-of-the-art DNA-editing procedures. Finally, we identify areas that warrant further investigation. Genetic manipulation of higher eukaryote cells plays a crucial role in basic and applied biology (Box 1). The advent and recent diversification of designer nuclease (DN) technologies (see Glossary) and their combination with nucleic acid and protein delivery systems have led to the emergence of a new field interchangeably dubbed genome engineering or GE. This biotechnology is becoming invaluable to not only interrogate but also efficiently rewrite DNA sequences in germ and somatic cells from an increasing number of organisms, including those of mammals [1Gaj T. et al.ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering.Trends Biotechnol. 2013; 31: 397-405Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 2Segal D.J. Meckler J.F. Genome engineering at the dawn of the golden age.Annu. Rev. Genomics Hum. Genet. 2013; 14: 135-158Crossref PubMed Scopus (29) Google Scholar]. Indeed, the universal role played by the genome in biological systems opens up the possibility for adapting the basic principles of GE to many disciplines and applications, including gene therapy, functional genomics, regenerative medicine, synthetic biology, and transgenesis.Box 1Classical genome modification technologiesThe genetic manipulation of mammalian cells can generically be achieved by non-targeted and targeted chromosomal integration of exogenously added recombinant DNA. The latter genetic engineering procedures are preferable over the former because they result in uniform transgene expression, owing to reduced chromosomal positional effects and predictable phenotypes, owing to decreased risk of endogenous gene disruption (i.e., insertional mutagenesis). However, until the late 1990s the deployment of such precise genome manipulations was restricted to particular HR-based experimental systems, most notably to those involving the generation of knock-in and knockout transgenic mice [99Capecchi M.R. Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century.Nat. Rev. Genet. 2005; 6: 507-512Crossref PubMed Scopus (218) Google Scholar]. In these systems, the very low HR rates and the high frequencies of random non-homologous chromosomal DNA insertions are circumvented by positive/negative selection regimens based on a combination of genetic tools and cytotoxic drugs. These strategies are, however, often difficult to apply in other biotechnological settings. Hence, early approaches aiming at genetic modification of mammalian somatic cells exploited instead the efficient, albeit non-targeted, chromosomal DNA integration capacity of γ-retroviral vectors (γ-RVs) [100Maetzig T. et al.Gammaretroviral vectors: biology, technology and application.Viruses. 2011; 3: 677-713Crossref PubMed Scopus (19) Google Scholar]. The γ-RV-mediated genetic modification of hematopoietic stem cells from boys afflicted by X-linked severe combined immunodeficiency provided the first proof-of-concept for gene therapy and, at the same time, materialized genotoxicity risks in the form of leukemogenesis in some of the treated patients [101Biasco L. et al.Retroviral integrations in gene therapy trials.Mol. Ther. 2012; 20: 709-716Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar]. These severe adverse events (SAEs) were linked to the insertion of γ-RV genomes carrying strong promoter/enhancer elements in the vicinity of proto-oncogenes [100Maetzig T. et al.Gammaretroviral vectors: biology, technology and application.Viruses. 2011; 3: 677-713Crossref PubMed Scopus (19) Google Scholar, 101Biasco L. et al.Retroviral integrations in gene therapy trials.Mol. Ther. 2012; 20: 709-716Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar]. These insertional mutagenesis findings initiated a trend towards HIV-1-based lentiviral vectors (LVs) [102Schambach A. et al.Biosafety features of lentiviral vectors.Hum. Gene Ther. 2013; 24: 132-142Crossref PubMed Scopus (22) Google Scholar] and the use of self-inactivating retroviral backbones in which viral regulatory sequences are replaced by more physiological cellular promoters [100Maetzig T. et al.Gammaretroviral vectors: biology, technology and application.Viruses. 2011; 3: 677-713Crossref PubMed Scopus (19) Google Scholar, 101Biasco L. et al.Retroviral integrations in gene therapy trials.Mol. Ther. 2012; 20: 709-716Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 102Schambach A. et al.Biosafety features of lentiviral vectors.Hum. Gene Ther. 2013; 24: 132-142Crossref PubMed Scopus (22) Google Scholar]. Of note, although LVs also display a semi-random integration profile, their proviral insertions are less biased towards the transcription start-sites of host cell genes [103Schröder A.R. et al.HIV-1 integration in the human genome favors active genes and local hotspots.Cell. 2002; 110: 521-529Abstract Full Text Full Text PDF PubMed Scopus (946) Google Scholar, 104Wu X. et al.Transcription start regions in the human genome are favored targets for MLV integration.Science. 2003; 300: 1749-1751Crossref PubMed Scopus (883) Google Scholar]. Furthermore, in contrast to γ-RVs, LVs possess active nuclear import mechanisms leading to efficient transduction of non-cycling cells [105Matreyek K.A. Engelman A. Viral and cellular requirements for the nuclear entry of retroviral preintegration nucleoprotein complexes.Viruses. 2013; 5: 2483-2511Crossref PubMed Scopus (8) Google Scholar]. Although genotoxic risks associated with LV-induced insertional mutagenesis remain [106Baum C. Data vs. dogma: HIV-1 integrations driving clonal selection.Mol Ther. 2014; 22: 1557-1558Abstract Full Text Full Text PDF PubMed Google Scholar], the aforementioned tangible improvements led to therapeutic outcomes in Wiskott–Aldrich syndrome and metachromatic leukodystrophy patients [107Aiuti A. et al.Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome.Science. 2013; 341: 1233151Crossref PubMed Scopus (116) Google Scholar, 108Biffi A. et al.Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy.Science. 2013; 341: 1233158Crossref PubMed Scopus (132) Google Scholar]. An alternative DNA modification approach consists of adapting transposon/transposase elements from vertebrate genomes. In contrast to retroviral vectors, some of these genetic mobile elements display a truly random chromosomal insertion profile – in other words, do not show a preference for gene bodies and associated regulatory sequences [109Ivics Z. et al.Transposon-mediated genome manipulation in vertebrates.Nat. Methods. 2009; 6: 415-422Crossref PubMed Scopus (109) Google Scholar]. The genetic manipulation of mammalian cells can generically be achieved by non-targeted and targeted chromosomal integration of exogenously added recombinant DNA. The latter genetic engineering procedures are preferable over the former because they result in uniform transgene expression, owing to reduced chromosomal positional effects and predictable phenotypes, owing to decreased risk of endogenous gene disruption (i.e., insertional mutagenesis). However, until the late 1990s the deployment of such precise genome manipulations was restricted to particular HR-based experimental systems, most notably to those involving the generation of knock-in and knockout transgenic mice [99Capecchi M.R. Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century.Nat. Rev. Genet. 2005; 6: 507-512Crossref PubMed Scopus (218) Google Scholar]. In these systems, the very low HR rates and the high frequencies of random non-homologous chromosomal DNA insertions are circumvented by positive/negative selection regimens based on a combination of genetic tools and cytotoxic drugs. These strategies are, however, often difficult to apply in other biotechnological settings. Hence, early approaches aiming at genetic modification of mammalian somatic cells exploited instead the efficient, albeit non-targeted, chromosomal DNA integration capacity of γ-retroviral vectors (γ-RVs) [100Maetzig T. et al.Gammaretroviral vectors: biology, technology and application.Viruses. 2011; 3: 677-713Crossref PubMed Scopus (19) Google Scholar]. The γ-RV-mediated genetic modification of hematopoietic stem cells from boys afflicted by X-linked severe combined immunodeficiency provided the first proof-of-concept for gene therapy and, at the same time, materialized genotoxicity risks in the form of leukemogenesis in some of the treated patients [101Biasco L. et al.Retroviral integrations in gene therapy trials.Mol. Ther. 2012; 20: 709-716Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar]. These severe adverse events (SAEs) were linked to the insertion of γ-RV genomes carrying strong promoter/enhancer elements in the vicinity of proto-oncogenes [100Maetzig T. et al.Gammaretroviral vectors: biology, technology and application.Viruses. 2011; 3: 677-713Crossref PubMed Scopus (19) Google Scholar, 101Biasco L. et al.Retroviral integrations in gene therapy trials.Mol. Ther. 2012; 20: 709-716Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar]. These insertional mutagenesis findings initiated a trend towards HIV-1-based lentiviral vectors (LVs) [102Schambach A. et al.Biosafety features of lentiviral vectors.Hum. Gene Ther. 2013; 24: 132-142Crossref PubMed Scopus (22) Google Scholar] and the use of self-inactivating retroviral backbones in which viral regulatory sequences are replaced by more physiological cellular promoters [100Maetzig T. et al.Gammaretroviral vectors: biology, technology and application.Viruses. 2011; 3: 677-713Crossref PubMed Scopus (19) Google Scholar, 101Biasco L. et al.Retroviral integrations in gene therapy trials.Mol. Ther. 2012; 20: 709-716Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 102Schambach A. et al.Biosafety features of lentiviral vectors.Hum. Gene Ther. 2013; 24: 132-142Crossref PubMed Scopus (22) Google Scholar]. Of note, although LVs also display a semi-random integration profile, their proviral insertions are less biased towards the transcription start-sites of host cell genes [103Schröder A.R. et al.HIV-1 integration in the human genome favors active genes and local hotspots.Cell. 2002; 110: 521-529Abstract Full Text Full Text PDF PubMed Scopus (946) Google Scholar, 104Wu X. et al.Transcription start regions in the human genome are favored targets for MLV integration.Science. 2003; 300: 1749-1751Crossref PubMed Scopus (883) Google Scholar]. Furthermore, in contrast to γ-RVs, LVs possess active nuclear import mechanisms leading to efficient transduction of non-cycling cells [105Matreyek K.A. Engelman A. Viral and cellular requirements for the nuclear entry of retroviral preintegration nucleoprotein complexes.Viruses. 2013; 5: 2483-2511Crossref PubMed Scopus (8) Google Scholar]. Although genotoxic risks associated with LV-induced insertional mutagenesis remain [106Baum C. Data vs. dogma: HIV-1 integrations driving clonal selection.Mol Ther. 2014; 22: 1557-1558Abstract Full Text Full Text PDF PubMed Google Scholar], the aforementioned tangible improvements led to therapeutic outcomes in Wiskott–Aldrich syndrome and metachromatic leukodystrophy patients [107Aiuti A. et al.Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome.Science. 2013; 341: 1233151Crossref PubMed Scopus (116) Google Scholar, 108Biffi A. et al.Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy.Science. 2013; 341: 1233158Crossref PubMed Scopus (132) Google Scholar]. An alternative DNA modification approach consists of adapting transposon/transposase elements from vertebrate genomes. In contrast to retroviral vectors, some of these genetic mobile elements display a truly random chromosomal insertion profile – in other words, do not show a preference for gene bodies and associated regulatory sequences [109Ivics Z. et al.Transposon-mediated genome manipulation in vertebrates.Nat. Methods. 2009; 6: 415-422Crossref PubMed Scopus (109) Google Scholar]. Various genetic engineering methodologies currently fall under the operative definition of GE, such as those based on site-specific recombinases (Box 2), single-stranded oligodeoxyribonucleotides (ssODNs), and recombinant adeno-associated viral vectors (rAAVs). However, we will focus on reviewing the strategies, parameters, and outcomes of GE procedures based on modifying target cell populations through the delivery of DNs, for which there is a growing and versatile portfolio (Box 3). DNs are built to generate double-stranded DNA breaks (DSBs) at predefined chromosomal positions and, in doing so, activate endogenous cellular DNA repair pathways. Indeed, the two main DNA repair pathways responsible for maintaining chromosomal integrity, non-homologous end-joining (NHEJ) and homologous recombination (HR), are activated by DSBs regardless of whether these lesions occur in a random or a site-specific fashion [3Lukacsovich T. et al.Repair of a specific double-strand break generated within a mammalian chromosome by yeast endonuclease I-SceI.Nucleic Acids Res. 1994; 22: 5649-5657Crossref PubMed Google Scholar, 4Rouet P. et al.Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 6064-6068Crossref PubMed Scopus (230) Google Scholar]. The repair of site-specific DSBs by NHEJ can create knockouts of either coding or cis-acting, non-coding sequences. These DSBs can also lead to knock-ins when repaired by HR events involving surrogate DSB repair substrates in the form of foreign donor DNA (Figure 1). Importantly, DNs can increase HR rates from 10−8 to 10−6 events per treated cell to frequencies as high as 1–30%. These high frequencies avoid the need for complex cell selection schemes in many experimental settings broadening, as a result, the applicability of HR-mediated GE.Box 2Recombinase- and homing endonuclease-assisted genome engineeringThe high demand for controlled chromosomal DNA insertion in both scientific and technological settings has been spurring the development of different genome manipulation technologies. In addition to artificial DNs, preeminent examples include the deployment of site-specific recombinases and integrases (e.g., CRE, FLP, and ϕC31) [110Turan S. Bode J. Site-specific recombinases: from tag-and-target- to tag-and-exchange-based genomic modifications.FASEB J. 2011; 25: 4088-4107Crossref PubMed Scopus (16) Google Scholar], adeno-associated virus (AAV) replicase/integrase complexes (i.e., Rep78/68) [111Recchia A. Mavilio F. Site-specific integration by the adeno-associated virus rep protein.Curr. Gene Ther. 2011; 11: 399-405Crossref PubMed Google Scholar, 112González-Prieto C. et al.HUH site-specific recombinases for targeted modification of the human genome.Trends Biotechnol. 2013; 31: 305-312Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar], and intron-encoded homing endonucleases (HEs), also known as meganucleases (e.g., I-SceI) [113Marcaida M.J. et al.Homing endonucleases: from basics to therapeutic applications.Cell. Mol. Life Sci. 2010; 67: 727-748Crossref PubMed Scopus (46) Google Scholar]. Although these native proteins are limited to targeting fixed chromosomal positions (e.g., ϕC31 and Rep78/68), and/or require the engineering of their cognate recognition sequences into the target cell DNA in the first place (e.g., CRE, FLP, and I-SceI), they have proved to be very useful tools in particular biotechnology platforms and experimental models. For instance, site-specific recombinases have been thoroughly used for setting up conditional gene activation/deletion systems [110Turan S. Bode J. Site-specific recombinases: from tag-and-target- to tag-and-exchange-based genomic modifications.FASEB J. 2011; 25: 4088-4107Crossref PubMed Scopus (16) Google Scholar, 114Gonçalves M.A.F.V. et al.Rapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity.PLoS ONE. 2010; 5: e10954Crossref PubMed Scopus (4) Google Scholar], whereas the I-SceI endonuclease has been instrumental in DSB repair studies [3Lukacsovich T. et al.Repair of a specific double-strand break generated within a mammalian chromosome by yeast endonuclease I-SceI.Nucleic Acids Res. 1994; 22: 5649-5657Crossref PubMed Google Scholar, 4Rouet P. et al.Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 6064-6068Crossref PubMed Scopus (230) Google Scholar]. In fact, the latter studies based on the generation of DSBs at specific model alleles and ensuing activation of DNA repair pathways provided a strong rationale for the development of sequence-tailored designer nucleases. This research, initiated in the 1990s with the introduction of ZFNs [115Kim Y.G. et al.Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 1156-1160Crossref PubMed Scopus (467) Google Scholar], heralded the beginning of the DN-assisted GE field. More recently, the tailoring of site-specific recombinases and rare-cutting HEs to new predefined target sequences is also underway. These technologies consist of designing chimeric proteins formed by recombinase or HE domains fused to DNA-binding motifs based on zinc-finger arrays or TALE repeats [116Gaj T. Barbas 3rd, C.F. Genome engineering with custom recombinases.Methods Enzymol. 2014; 546: 79-91Crossref PubMed Google Scholar]. In addition, strategies based on complex protein engineering endeavors aiming at altering HE target-site preference have equally been pursued [117Redondo P. et al.Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases.Nature. 2008; 456: 107-111Crossref PubMed Scopus (92) Google Scholar].Box 3Characteristics and modus operandi of the main classes of DNsZFNs (Figure IA ) are modular artificial proteins consisting of an array of typically 4–6 synthetic Cys2-His2 zinc-finger motifs fused through a linker to the catalytic domain of a restriction enzyme, traditionally that of the type IIS endonuclease FokI (the recognition and cleavage DNA sequences of type IIS restriction enzymes are non-overlapping). Functional ZFNs consist of two monomers assembled in a tail-to-tail orientation at the target site on opposite DNA strands. The local dimerization of the FokI nuclease domains catalyzes upper and lower strand DNA cleavage at a spacer sequence located between the ZFN half-target sites. As a result, a site-specific DSB is formed.TALENs (Figure IB) display an architecture generically similar to that of ZFNs because the DNA-binding domain (DBD) is also fused via a linker to the non-specific FokI nuclease domain that becomes catalytically active upon in situ dimerization. For TALENs, however, the DBD is derived from TALE proteins found in specific phytopathogenic bacteria (e.g., genus Xanthomomas) and comprises an array of typically 15.5–19.5 repeat units of approximately 34 residues each. The repeat residues at positions 12 and 13, called repeat-variable di-residues (RVDs), dictate nucleotide recognition (e.g., RVDs NI, NG, and HD recognize preferentially A, T, and C, respectively). Commonly used TALEN scaffolds comprise 17.5 repeats per monomer and are encoded by ORFs of approximately 3 kb.RGNs (Figure IC) are RNA-dependent nucleases built on components from clustered, regularly interspaced, short palindromic repeat (CRISPR)-associated Cas systems evolved in bacteria and archaea as immune mechanisms against foreign nucleic acids. The most commonly used RGNs are based on the type II CRISPR-Cas9 nuclease system from Streptococcus pyogenes. Cas9 is a large protein (∼160 kDa) encoded by a 4.1 kb ORF and contains two nuclease domains (RuvC and HNH). This nuclease is addressed to the target site via its association with a single guide RNA (gRNA) molecule. The gRNA component is a bipartite molecule engineered by fusing a sequence-tailored CRISPR RNA (crRNA) to a scaffolding transactivating crRNA (tracrRNA). The target site consists of a nucleotide stretch matching the 5′ terminal gRNA sequence (usually 20 bp in length) followed by a short nucleotide sequence called protospacer adjacent motif (PAM; NGG, in the case of S. pyogenes Cas9). The fact that target sequence specificity of RGNs is governed by RNA–DNA hybridization, as opposed to protein–DNA interactions, confers versatility and multiplexing capabilities to RGNs.Figure 1Illustration of the main GE strategies based on DNs. (A) NHEJ-mediated GE. Site-specific DSBs (cyan arrowheads) activate NHEJ repair pathways. These processes can be exploited for obtaining different GE outcomes. Left panel, site-specific DSBs can yield reading-frame shifts resulting from indels (red boxes) that disrupt and restore, respectively, in-frame and out-of-frame sequences. Middle panel, simultaneous induction of tandem DSBs by DN pairs (multiplexing) can result in the deletion of the intervening sequence following end-to-end ligation of the distal chromosomal breakpoints. Alternatively, re-insertion of the intervening sequence in an ‘antisense’ orientation can also occur (not drawn). Of note, indel formation is, in this case, less likely because the newly formed junction (j) should yield a sequence that is not susceptible to DN activity. Right panel, DN multiplexing applied to sequences in different chromosomes can be exploited for studying well-defined translocations. (B) HR-mediated GE. Site-specific DSBs can also activate the HR pathway. In the presence of foreign DNA flanked by nucleotide sequences identical to those framing the target site (donor DNA) the HR process yields well-defined junctions between the endogenous and the chromosomally integrated exogenous DNA. By using judiciously constructed donor DNA templates, this DN-induced homology-directed gene targeting can be exploited to introduce or remove specific mutations or single-nucleotide polymorphisms (asterisks) or, alternatively, insert a gene tag (not drawn), a complementary DNA (not drawn), or an entire transgene at a predefined chromosomal position (e.g., a ‘safe harbor’ whose prototypic example is that of the AAVS1 locus on the human chromosome 19 at 19q13.42). Solid boxes and continuous lines represent exons and introns, respectively. Abbreviations: AAVS1, adeno-associated virus integration site 1; DSB, double-stranded DNA break; DN, designer nuclease; GE, genome editing; HR, homologous recombination; NHEJ, non-homologous end-joining.View Large Image Figure ViewerDownload (PPT) The high demand for controlled chromosomal DNA insertion in both scientific and technological settings has been spurring the development of different genome manipulation technologies. In addition to artificial DNs, preeminent examples include the deployment of site-specific recombinases and integrases (e.g., CRE, FLP, and ϕC31) [110Turan S. Bode J. Site-specific recombinases: from tag-and-target- to tag-and-exchange-based genomic modifications.FASEB J. 2011; 25: 4088-4107Crossref PubMed Scopus (16) Google Scholar], adeno-associated virus (AAV) replicase/integrase complexes (i.e., Rep78/68) [111Recchia A. Mavilio F. Site-specific integration by the adeno-associated virus rep protein.Curr. Gene Ther. 2011; 11: 399-405Crossref PubMed Google Scholar, 112González-Prieto C. et al.HUH site-specific recombinases for targeted modification of the human genome.Trends Biotechnol. 2013; 31: 305-312Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar], and intron-encoded homing endonucleases (HEs), also known as meganucleases (e.g., I-SceI) [113Marcaida M.J. et al.Homing endonucleases: from basics to therapeutic applications.Cell. Mol. Life Sci. 2010; 67: 727-748Crossref PubMed Scopus (46) Google Scholar]. Although these native proteins are limited to targeting fixed chromosomal positions (e.g., ϕC31 and Rep78/68), and/or require the engineering of their cognate recognition sequences into the target cell DNA in the first place (e.g., CRE, FLP, and I-SceI), they have proved to be very useful tools in particular biotechnology platforms and experimental models. For instance, site-specific recombinases have been thoroughly used for setting up conditional gene activation/deletion systems [110Turan S. Bode J. Site-specific recombinases: from tag-and-target- to tag-and-exchange-based genomic modifications.FASEB J. 2011; 25: 4088-4107Crossref PubMed Scopus (16) Google Scholar, 114Gonçalves M.A.F.V. et al.Rapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity.PLoS ONE. 2010; 5: e10954Crossref PubMed Scopus (4) Google Scholar], whereas the I-SceI endonuclease has been instrumental in DSB repair studies [3Lukacsovich T. et al.Repair of a specific double-strand break generated within a mammalian chromosome by yeast endonuclease I-SceI.Nucleic Acids Res. 1994; 22: 5649-5657Crossref PubMed Google Scholar, 4Rouet P. et al.Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 6064-6068Crossref PubMed Scopus (230) Google Scholar]. In fact, the latter studies based on the generation of DSBs at specific model alleles and ensuing activation of DNA repair pathways provided a strong rationale for the development of sequence-tailored designer nucleases. This research, initiated in the 1990s with the introduction of ZFNs [115Kim Y.G. et al.Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 1156-1160Crossref PubMed Scopus (467) Google Scholar], heralded the beginning of the DN-assisted GE field. More recently, the tailoring of site-specific recombinases and rare-cutting HEs to new predefined target sequences is also underway. These technologies consist of designing chimeric proteins formed by recombinase or HE domains fused to DNA-binding motifs based on zinc-finger arrays or TALE repeats [116Gaj T. Barbas 3rd, C.F. Genome engineering with custom recombinases.Methods Enzymol. 2014; 546: 79-91Crossref PubMed Google Scholar]. In addition, strategies based on complex protein engineering endeavors aiming at altering HE target-site preference have equally been pursued [117Redondo P. et al.Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases.Nature. 2008; 456: 107-111Crossref PubMed Scopus (92) Google Scholar]. ZFNs (Figure IA ) are modular artificial proteins consisting of an array of typically 4–6 synthetic Cys2-His2 zinc-finger motifs fused through a linker to the catalytic domain of a restriction enzyme, traditionally that of the type IIS endonuclease FokI (the recognition and cleavage DNA sequences of type IIS restriction enzymes are non-overlapping). Functional ZFNs consist of two monomers assembled in a tail-to-tail orientation at the target site on opposite DNA strands. The local dimerization of the FokI nuclease domains catalyzes upper and lower strand DNA cleavage at a spacer sequence located between the ZFN half-target sites. As a result, a site-specific DSB is formed. TALENs (Figure IB) display an architecture generically similar to that of ZFNs because the DNA-binding domain (DBD) is also fused via a linker to the non-specific FokI nuclease domain that becomes catalytically active upon in situ dimerization. For TALENs, however, the DBD is derived from TALE proteins found in specific phytopathogenic bacteria (e.g., genus Xanthomomas) and comprises an array of typically 15.5–19.5 repeat units of approximately 34 residues each. The repeat residues at positions 12 and 13, called repeat-variable di-residues (RVDs), dictate nucleotide recognition (