Unraveling DNA Triplex Assembly: Mass Spectrometric Investigation of Modified Triplex Forming Oligonucleotides for Enhanced Gene Targeting.

Deoxyribonucleic acid triplexes have potential roles in a range of biological processes involving gene and transcriptional regulation. A major challenge in exploiting the formation of these higher-order structures to target genes in vivo is their low stability, which is dependent on many factors including the length and composition of bases in the sequence. Here, different DNA base modifications have been explored, primarily using native mass spectrometry, in efforts to enable stronger binding between the triplex forming oligonucleotide (TFO) and duplex target sites. These modifications can also be used to overcome pyrimidine interruptions in the duplex sequence in promoter regions of genomes, to expand triplex target sequences for antigene therapies. Using model sequences with a single pyrimidine interruption, triplex forming oligonucleotides containing locked nucleic acid base modifications were shown to have a higher triplex binding propensity than DNA-only and dSpacer-containing TFOs. However, the triplex forming ability of these systems was limited by the competitive formation of multiple higher order assemblies. Triplex forming sequences that correspond to specific gene targets from the Pseudomonas aeruginosa genome were also investigated, with LNA-containing TFOs the only variant able to form triplex using these sequences. This work indicates the advantages of utilizing synthetically modified TFOs to form triplex assemblies in vivo for potential therapeutic applications and highlights the advantages of native mass spectrometry for the study of their formation.

Recent Progress in Gene-Targeting Therapies for Spinal Muscular Atrophy: Promises and Challenges.

Spinal muscular atrophy (SMA) is a severe genetic disorder characterized by the loss of motor neurons, leading to progressive muscle weakness, loss of mobility, and respiratory complications. In its most severe forms, SMA can result in death within the first two years of life if untreated. The condition arises from mutations in the SMN1 (survival of motor neuron 1) gene, causing a deficiency in the survival motor neuron (SMN) protein. Humans possess a near-identical gene, SMN2, which modifies disease severity and is a primary target for therapies. Recent therapeutic advancements include antisense oligonucleotides (ASOs), small molecules targeting SMN2, and virus-mediated gene replacement therapy delivering a functional copy of SMN1. Additionally, recognizing SMA's broader phenotype involving multiple organs has led to the development of SMN-independent therapies. Evidence now indicates that SMA affects multiple organ systems, suggesting the need for SMN-independent treatments along with SMN-targeting therapies. No single therapy can cure SMA; thus, combination therapies may be essential for comprehensive treatment. This review addresses the SMA etiology, the role of SMN, and provides an overview of the rapidly evolving therapeutic landscape, highlighting current achievements and future directions.

All-RNA-mediated targeted gene integration in mammalian cells with rationally engineered R2 retrotransposons.

All-RNA-mediated targeted gene integration methods, rendering reduced immunogenicity, effective deliverability with non-viral vehicles, and a low risk of random mutagenesis, are urgently needed for next-generation gene addition technologies. Naturally occurring R2 retrotransposons hold promise in this context due to their site-specific integration profile. Here, we systematically analyzed the biodiversity of R2 elements and screened several R2 orthologs capable of full-length gene insertion in mammalian cells. Robust R2 system gene integration efficiency was attained using combined donor RNA and protein engineering. Importantly, the all-RNA-delivered engineered R2 system showed effective integration activity, with efficiency over 60% in mouse embryos. Unbiased high-throughput sequencing demonstrated that the engineered R2 system exhibited high on-target integration specificity (99%). In conclusion, our study provides engineered R2 tools for applications based on hit-and-run targeted DNA integration and insights for further optimization of retrotransposon systems.

Cas12a-mediated gene targeting by sequential transformation strategy in Arabidopsis thaliana.

Gene targeting (GT) allows precise manipulation of genome sequences, such as knock-ins and sequence substitutions, but GT in seed plants remains a challenging task. Engineered sequence-specific nucleases (SSNs) are known to facilitate GT via homology-directed repair (HDR) in organisms. Here, we demonstrate that Cas12a and a temperature-tolerant Cas12a variant (ttCas12a) can efficiently establish precise and heritable GT at two loci in Arabidopsis thaliana (Arabidopsis) through a sequential transformation strategy. As a result, ttCas12a showed higher GT efficiency than unmodified Cas12a. In addition, the efficiency of transcriptional and translational enhancers for GT via sequential transformation strategy was also investigated. These enhancers and their combinations were expected to show an increase in GT efficiency in the sequential transformation strategy, similar to previous reports of all-in-one strategies, but only a maximum twofold increase was observed. These results indicate that the frequency of double strand breaks (DSBs) at the target site is one of the most important factors determining the efficiency of genetic GT in plants. On the other hand, a higher frequency of DSBs does not always lead to higher efficiency of GT, suggesting that some additional factors are required for GT via HDR. Therefore, the increase in DSB can no longer be expected to improve GT efficiency, and a new strategy needs to be established in the future. This research opens up a wide range of applications for precise and heritable GT technology in plants.

Construction of an expression platform for fungal secondary metabolite biosynthesis in Penicillium crustosum.

Filamentous fungi are prolific producers of bioactive natural products and play a vital role in drug discovery. Yet, their potential cannot be fully exploited since many biosynthetic genes are silent or cryptic under laboratory culture conditions. Several strategies have been applied to activate these genes, with heterologous expression as one of the most promising approaches. However, successful expression and identification of new products are often hindered by host-dependent factors, such as low gene targeting efficiencies, a high metabolite background, or a lack of selection markers. To overcome these challenges, we have constructed a Penicillium crustosum expression host in a pyrG deficient strain by combining the split-marker strategy and CRISPR-Cas9 technology. Deletion of ligD and pcribo improved gene targeting efficiencies and enabled the use of an additional selection marker in P. crustosum. Furthermore, we reduced the secondary metabolite background by inactivation of two highly expressed gene clusters and abolished the formation of the reactive ortho-quinone methide. Finally, we replaced the P. crustosum pigment gene pcr4401 with the commonly used Aspergillus nidulans wA expression site for convenient use of constructs originally designed for A. nidulans in our P. crustosum host strain. As proof of concept, we successfully expressed a single polyketide synthase gene and an entire gene cluster at the P. crustosum wA locus. Resulting transformants were easily detected by their albino phenotype. With this study, we provide a highly efficient platform for heterologous expression of fungal genes. KEY POINTS: Construction of a highly efficient Penicillium crustosum heterologous expression host Reduction of secondary metabolite background by genetic dereplication strategy Integration of wA site to provide an alternative host besides Aspergillus nidulans.

Targeted insertion of conditional expression cassettes into the mouse genome using the modified i-PITT.

BACKGROUND: Transgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called "pronuclear injection-based targeted transgenesis" (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic LoxP sites or attP sites) to create seed mice and then injecting Cre recombinase or PhiC31 integrase mRNAs along with a compatible donor plasmid into zygotes derived from the seed mice. PITT and its subsequent version, improved PITT (i-PITT), overcome disadvantages of conventional Tg mice such as lack of consistent and reliable expression of the cassettes among different Tg mouse lines, and the PITT approach is superior in terms of cost and labor. One of the limitations of PITT, particularly using Cre-mRNA, is that the approach cannot be used for insertion of conditional expression cassettes using Cre-LoxP site-specific recombination. This is because the LoxP sites in the donor plasmids intended for achieving conditional expression of the transgene will interfere with the PITT recombination reaction with LoxP sites in the landing pad. RESULTS: To enable the i-PITT method to insert a conditional expression cassette, we modified the approach by simultaneously using PhiC31o and FLPo mRNAs. We demonstrate the strategy by creating a model containing a conditional expression cassette at the Rosa26 locus with an efficiency of 13.7%. We also demonstrate that inclusion of FLPo mRNA excludes the insertion of vector backbones in the founder mice. CONCLUSIONS: Simultaneous use of PhiC31 and FLP in i-PITT approach allows insertion of donor plasmids containing Cre-loxP-based conditional expression cassettes.

In Vitro Amplification and Selection of Engineered RNase P Ribozyme for Gene Targeting Applications.

Ribozymes engineered from the RNase P catalytic RNA (M1 RNA) represent promising gene-targeting agents for clinical applications. We describe in this report an in vitro amplification and selection procedure for generating active RNase P ribozyme variants with improved catalytic efficiency. Using the amplification and selection procedure, we have previously generated ribozyme variants that were highly active in cleaving a herpes simplex virus 1-encoded mRNA in vitro and inhibiting its expression in virally infected human cells. In this chapter, we use an overlapping region of the mRNAs for the IE1 and IE2 proteins of human cytomegalovirus (HCMV) as a target substrate. We provide detailed protocols and include methods for establishing the procedure for the amplification and selection of active mRNA-cleaving RNase P ribozymes. The in vitro amplification and selection system represents an excellent approach for engineering highly active RNase P ribozymes that can be used in both basic research and clinical applications.

Generation of Rare Human NMDA Receptor Variants in Mice.

The analysis of rare NMDAR gene variants in mice, coupled with a fundamental understanding of NMDAR function, plays a crucial role in achieving therapeutic success when addressing NMDAR dysfunctions in human patients. For the generation of such NMDAR mouse models, a basic knowledge of receptor structure, along with skills in database sequence analysis, cloning in E. coli, genetic manipulation of embryonic stem (ES) cells, and ultimately the genetic modification of mouse embryos, is essential. Primarily, this chapter will focus on the design and synthesis of NMDAR gene-targeting vectors that can be used successfully for the genetic manipulation of mice. We will outline the core principles of the design and synthesis of a gene targeting vector that facilitates the introduction of single-point mutations in NMDAR-encoding genes in mice. The transformation of ES cells, selection of positive ES cell colonies, manipulation of mouse embryos, and genotyping strategies will be described briefly.

Generation of FOXJ1-EGFP knock-in reporter human embryonic stem cell line, WAe001-A-2D, using CRISPR/Cas9-based gene targeting.

Forkhead box protein J1 (FOXJ1), a member of the forkhead family, is an important transcription factor regulating multiciliated cell differentiation and motile ciliogenic program. Here, we established a FOXJ1- EGFP knock-in human embryonic stem cell (hESC) line by inserting a P2A-EGFP gene cassette of FOXJ1 using CRISPR/Cas9 system. The reporter cell line retained a normal karyotype, expressed comparable pluripotent marker genes, and maintained differentiation potential. This reporter cell line enables live identification of multiciliated cells during the general lung differentiation and will be a valuable tool for studying the multiciliated cell differentiation, ciliogenesis and mechanism of related pulmonary diseases.

Novel off-Targeting Events Identified after Genome Wide Analysis of CRISPR-Cas Edited Pigs.

CRISPR-Cas technology has transformed our ability to introduce targeted modifications, allowing unconventional animal models such as pigs to model human diseases and improve its value for food production. The main concern with using the technology is the possibility of introducing unwanted modifications in the genome. In this study, we illustrate a pipeline to comprehensively identify off-targeting events on a global scale in the genome of three different gene-edited pig models. Whole genome sequencing paired with an off-targeting prediction software tool filtered off-targeting events amongst natural variations present in gene-edited pigs. This pipeline confirmed two known off-targeting events in IGH knockout pigs, AR and RBFOX1, and identified other presumably off-targeted loci. Independent validation of the off-targeting events using other gene-edited DNA confirmed two novel off-targeting events in RAG2/IL2RG knockout pig models. This unique strategy offers a novel tool to detect off-targeting events in genetically heterogeneous species after genome editing.

Gene targeting in adult organs using in vivo cleavable donor plasmids for CRISPR-Cas9 and CRISPR-Cas12a.

The CRISPR-Cas system for in vivo genome editing is a powerful tool for gene therapy against several diseases. We have previously developed the pCriMGET_9-12a system, an in vivo cleavable donor plasmid for precise targeted knock-in of exogenous DNA by both Cas9 and Cas12a. Here, we show that the pCriMGET_9-12a system can be applied for in vivo in-frame knock-in of exogenous DNA in adult mouse liver by hydrodynamic delivery of the targeting plasmids. The in vivo cleavable pCriMGET_9-12a donor plasmids significantly increased the knock-in efficiency of both CRISPR-Cas9 and CRISPR-Cas12a in the adult mouse liver compared to uncleavable donor plasmids. This strategy also achieved in-frame reporter gene knock-in without indel mutations. Therefore, in vivo gene targeting using the pCriMGET_9-12a system may contribute to the establishment of safer, more precise, versatile and efficient gene therapy methods in adult organs.

Molecular analysis of the role of polymerase theta in gene targeting in Arabidopsis thaliana.

Precise genetic modification can be achieved via a sequence homology-mediated process known as gene targeting (GT). Whilst established for genome engineering purposes, the application of GT in plants still suffers from a low efficiency for which an explanation is currently lacking. Recently reported reduced rates of GT in A. thaliana deficient in polymerase theta (Polθ), a core component of theta-mediated end joining (TMEJ) of DNA breaks, have led to the suggestion of a direct involvement of this enzyme in the homology-directed process. Here, by monitoring homology-driven gene conversion in plants with CRISPR reagent and donor sequences pre-integrated at random sites in the genome (in planta GT), we demonstrate that Polθ action is not required for GT, but instead suppresses the process, likely by promoting the repair of the DNA break by end-joining. This finding indicates that lack of donor integration explains the previously established reduced GT rates seen upon transformation of Polθ-deficient plants. Our study additionally provides insight into ectopic gene targeting (EGT), recombination events between donor and target that do not map to the target locus. EGT, which occurs at similar frequencies as "true" GT during transformation, was rare in our in planta GT experiments arguing that EGT predominantly results from target locus recombination with nonintegrated T-DNA molecules. By describing mechanistic features of GT our study provides directions for the improvement of precise genetic modification of plants.

Application of multiple sgRNAs boosts efficiency of CRISPR/Cas9-mediated gene targeting in Arabidopsis.

BACKGROUND: Precise gene targeting (GT) is a powerful tool for heritable precision genome engineering, enabling knock-in or replacement of the endogenous sequence via homologous recombination. We recently established a CRISPR/Cas9-mediated approach for heritable GT in Arabidopsis thaliana (Arabidopsis) and rice and reported that the double-strand breaks (DSBs) frequency of Cas9 influences the GT efficiency. However, the relationship between DSBs and GT at the same locus was not examined. Furthermore, it has never been investigated whether an increase in the number of copies of sgRNAs or the use of multiple sgRNAs would improve the efficiency of GT. RESULTS: Here, we achieved precise GT at endogenous loci Embryo Defective 2410 (EMB2410) and Repressor of Silencing 1 (ROS1) using the sequential transformation strategy and the combination of sgRNAs. We show that increasing of sgRNAs copy number elevates both DSBs and GT efficiency. On the other hand, application of multiple sgRNAs does not always enhance GT efficiency. Our results also suggested that some inefficient sgRNAs would play a role as a helper to facilitate other sgRNAs DSBs activity. CONCLUSIONS: The results of this study clearly show that DSB efficiency, rather than mutation pattern, is one of the most important key factors determining GT efficiency. This study provides new insights into the relationship between sgRNAs, DSBs, and GTs and the molecular mechanisms of CRISPR/Cas9-mediated GTs in plants.

CRISPR/Cas9 system is a suitable gene targeting editing tool to filamentous fungus Monascus pilosus.

Monascus pilosus has been used to produce lipid-lowering drugs rich in monacolin K (MK) for a long period. Genome mining reveals there are still many potential genes worth to be explored in this fungus. Thereby, efficient genetic manipulation tools will greatly accelerate this progress. In this study, we firstly developed the protocol to prepare protoplasts for recipient of CRISPR/Cas9 system. Subsequently, the vector and donor DNA were co-transformed into recipients (106 protoplasts/mL) to produce 60-80 transformants for one test. Three genes (mpclr4, mpdot1, and mplig4) related to DNA damage response (DDR) were selected to compare the gene replacement frequencies (GRFs) of Agrobacterium tumefaciens-mediated transformation (ATMT) and CRISPR/Cas9 gene editing system (CGES) in M. pilosus MS-1. The results revealed that GRF of CGES was approximately five times greater than that of ATMT, suggesting that CGES was superior to ATMT as a targeting gene editing tool in M. pilosus MS-1. The inactivation of mpclr4 promoted DDR via the non-homologous end-joining (NHEJ) and increased the tolerances to DNA damaging agents. The inactivation of mpdot1 blocked DDR and led to the reduced tolerances to DNA damaging agents. The inactivation of mplig4 mainly blocked the NHEJ pathway and led to obviously reduced tolerances to DNA damaging agents. The submerged fermentation showed that the ability to produce MK in strain Δmpclr4 was improved by 52.6% compared to the wild type. This study provides an idea for more effective exploration of gene functions in Monascus strains. KEY POINTS: • A protocol of high-quality protoplasts for CGES has been developed in M. pilosus. • The GRF of CGES was about five times that of ATMT in M. pilosus. • The yield of MK for Δmpclr4 was enhanced by 52.6% compared with the wild type.

Mouse Gene-Targeting Strategies for Maximum Ease and Versatility.

Well-planned strategies are an essential prerequisite for any mutational analysis involving gene targeting. Consideration of the advantages or disadvantages of different methods will aid in the production of a final product that is both technically feasible and versatile. Strategies for gene-targeting experiments in the mouse are discussed, including the rationale behind some of the common elements of gene-targeting vectors, such as homologous DNA and the use of different site-specific recombinases. We detail positive and negative selection as well as screening strategies for homologous recombination events in embryonic stem (ES) cells. For the planning stages of making different types of alleles, we first consider general strategies and then provide details specific to either homologous recombination in ES cells or making alleles by gene editing with CRISPR-Cas in preimplantation embryos. The types of alleles considered are null or knockout alleles, reporter gene knock-in alleles, point mutations, and conditional null alleles.

Versatile generation of precise gene edits in bovines using SEGCPN.

BACKGROUND: Gene knockout and knock-in have been widely performed in large farm animals based on genome editing systems. However, many types of precise gene editing, including targeted deletion, gene tagging, and large gene fragment replacement, remain a challenge in large farm animals. RESULTS: Here, we established versatile self-excising gene-targeting technology in combination with programmable nucleases (SEGCPN) to efficiently generate various types of precise gene editing in bovine. First, we used this versatile method to successfully generate bovine embryos with point mutations and 11-bp deletions at the MSTN locus. Second, we successfully generated bulls with EGFP labeling at the SRY locus. Finally, we successfully generated humanized cows in which the endogenous 18-kb α-casein gene was replaced with a 2.6-kb human α-lactalbumin gene. CONCLUSIONS: In summary, our new SEGCPN method offers unlimited possibilities for various types of precise gene editing in large animals for application both in agriculture and disease models.

Scarless Modification of the Drosophila Genome Near Any Mapped attP Sites.

Here we describe a Drosophila genome engineering technique that can scarlessly modify genomic sequences near any mapped attP attachment site previously integrated by transposon mobilization or gene targeting. This technique combines two highly efficient and robust procedures: phiC31 integrase-mediated site-specific integration and homing endonuclease-mediated resolution of local duplications. In this technique, a donor fragment containing the desired mutation(s) is first integrated into a selected attP site near the target locus by phiC31 integrase-mediated site-specific integration, which creates local duplications consisting of the mutant-containing donor fragment and the wild-type target locus. Next, homing endonuclease-induced double-stranded DNA breaks trigger recombination between the duplications and resolve the target locus to generate scarless mutant alleles. In every step, the desired flies can be easily identified by patterns of dominant markers, so no large-scale screens are needed. This technique is highly efficient and can be used to generate scarless point mutations, insertions, and deletions. The availability of large libraries of mapped attP site-containing transposon/CRISPR insertions in Drosophila allows the modification of more than half of the euchromatic Drosophila genome at a high efficiency. As more and more attP-containing insertions are generated and mapped, this technique will be able to modify larger portions of the Drosophila genome. The principles of this technique are applicable to other organisms where modifications to the genome are feasible. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Verifying attP-containing insertions Support Protocol: Extracting genomic DNA Basic Protocol 2: Generating the donor plasmid Basic Protocol 3: Injecting the donor plasmid and establishing transformant stocks Basic Protocol 4: Verifying the transformants Basic Protocol 5: Generating the final scarless alleles Basic Protocol 6: Verifying the final alleles.

Immune-check blocking combination multiple cytokines shown curative potential in mice tumor model.

OBJECTIVE: In order to ensure the stable transcription of target genes, we constructed a eukaryotic high expression vector carrying an immune-check inhibitor PD-1v and a variety of cytokines, and studied their effects on activating immune response to inhibit tumor growth. METHODS: A novel eukaryotic expression plasmid vector named pT7AMPCE containing T7RNA polymerase, T7 promoter, internal ribosome entry site (IRES), and poly A tailing signal was constructed by T4 DNA ligase, on which homologous recombination was used to clone and construct the vector carrying PD-1v, IL-2/15, IL-12, GM-CSF, and GFP. In vitro transfection of CT26 cells was performed, and the protein expression of PD-1v, IL-12 and GM-CSF was detected by Western blot and ELISA after 48 h. Mice were subcutaneously inoculated with CT26-IRFP tumor cells in the rib abdomen, and the tumor tissues were injected with PD-1v, IL-2/15, IL-12, and GM-CSF recombinant plasmids for treatment during the experimental period. The efficacy of the treatment was evaluated by assay tumor size and survival time of tumor-bearing mice during the experiment. Expression levels of IFN-γ, TNF, IL-4, IL-2, and IL-5 in mouse blood were measured using the CBA method. Tumor tissues were extracted and immune cell infiltration in tumor tissues was detected by HE staining and the IHC method. RESULTS: The recombinant plasmids carrying PD-1v, IL-2/15, IL-12, and GM-CSF were successfully constructed, and the Western blot and ELISA results showed that PD-1v, IL-12, and GM-CSF were expressed in the supernatant of CT26 cells 48 h after in vitro cell transfection. The combined application of PD-1v, IL-2/15, IL-12, and GM-CSF recombinant plasmids significantly inhibited tumor growth in mice, and the tumor growth rate was significantly lower than that in the blank control group and GFP plasmid control group (p < 0.05). Cytometric bead array data suggested that the combination of PD-1v and various cytokines can effectively activate immune cells. HE and IHC analysis revealed plenty of immune cell infiltrates in the tumor tissue, and a large proportion of tumor cells showed the necrotic phenotype in the combination treatment group. CONCLUSION: The combination of immune check blockade and multiple cytokine therapy can significantly activate the body's immune response and inhibit tumor growth.

High-efficient CRISPR/Cas9-mediated gene targeting to establish cell models of ciliopathies.

Primary cilia are antenna-like structures developed on the cell surface of mammalian cells during the quiescent G0 phase. Primary cilia in mammalian cells receive extracellular signals for early development and cell tissue homeostasis. Ciliopathies characterized with congenital anomalies such as cerebellar hypoplasia, polycystic kidney and polydactyly are caused by germline mutations of ciliary structure- and function-related genes. Gene knock-out techniques in ciliated cultured cells with the uniformed genetic background are useful to evaluate the pathophysiological roles of ciliopathy-related gene products. Genome editing technology has been applied into the gene knock-out in many types of cultured cell lines. However, the frequency of genome editing varies according to cell species and cycle because of dependency on error-free homology-directed repair (HDR) activity. The human telomerase reverse transcriptase-immortalized retinal pigmented epithelial cell line (hTERT-RPE1) is well known for its suitability in cilia research. However, the efficacy of the HDR-mediated knock-out clone isolation was low. Here, we introduce the clustered regularly interspaced short palindromic repeats-obligate ligation-gated recombination (CRISPR-ObLiGaRe) system, which is a nonhomologous end-joining (NHEJ)-mediated gene targeting method, to generate the knock-out clones effectively even in the lower-HDR activity cell lines including hTERT-RPE1 cell. This CRISPR-ObLiGaRe system is a powerful tool for establishing ciliopathy model cell libraries and identifying each gene function in cilia-related phenotypes.

Transgenesis and Genome Engineering: A Historical Review.

Our ability to modify DNA molecules and to introduce them into mammalian cells or embryos almost appears in parallel, starting from the 1970s of the last century. Genetic engineering techniques rapidly developed between 1970 and 1980. In contrast, robust procedures to microinject or introduce DNA constructs into individuals did not take off until 1980 and evolved during the following two decades. For some years, it was only possible to add transgenes, de novo, of different formats, including artificial chromosomes, in a variety of vertebrate species or to introduce specific mutations essentially in mice, thanks to the gene-targeting methods by homologous recombination approaches using mouse embryonic stem (ES) cells. Eventually, genome-editing tools brought the possibility to add or inactivate DNA sequences, at specific sites, at will, irrespective of the animal species involved. Together with a variety of additional techniques, this chapter will summarize the milestones in the transgenesis and genome engineering fields from the 1970s to date.