Gene Editing on Center Stage

Targeting DNA With Fingers and TALENs.

Genome editing is a relevant, versatile, and preferred tool for crop improvement, as well as for functional genomics. In this review, we summarize the advances in gene-editing techniques, such as zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) associated with the Cas9 and Cpf1 proteins. These tools support great opportunities for the future development of plant science and rapid remodeling of crops. Furthermore, we discuss the brief history of each tool and provide their comparison and different applications. Among the various genome-editing tools, CRISPR has become the most popular; hence, it is discussed in the greatest detail. CRISPR has helped clarify the genomic structure and its role in plants: For example, the transcriptional control of Cas9 and Cpf1, genetic locus monitoring, the mechanism and control of promoter activity, and the alteration and detection of epigenetic behavior between single-nucleotide polymorphisms (SNPs) investigated based on genetic traits and related genome-wide studies. The present review describes how CRISPR/Cas9 systems can play a valuable role in the characterization of the genomic rearrangement and plant gene functions, as well as the improvement of the important traits of field crops with the greatest precision. In addition, the speed editing strategy of gene-family members was introduced to accelerate the applications of gene-editing systems to crop improvement. For this, the CRISPR technology has a valuable advantage that particularly holds the scientist’s mind, as it allows genome editing in multiple biological systems.

Targeted nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However, realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids, the direct delivery of nuclease proteins to cells provides rapid action and fast turnover, leading to fewer off-target effects while maintaining high rates of targeted modification. These features make nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression, purification and delivery of ZFN proteins, which are intrinsically cell-permeable; TALEN proteins, which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein, whose nucleofection into cells facilitates rapid induction of multiplexed modifications, are described, along with procedures for evaluating nuclease protein activity. Once they are constructed, nuclease proteins can be expressed and purified within 6 d, and they can be used to induce genomic modifications in human cells within 2 d.

Application of the CRISPR–Cas System for Efficient Genome Engineering in Plants

Transcription activator-like (TAL) effector nucleases (TALENs) have enabled the introduction of targeted genetic alterations into a broad range of cell lines and organisms. These customizable nucleases are comprised of programmable sequence-specific DNA-binding modules derived from TAL effector proteins fused to the non-specific FokI cleavage domain. Delivery of these nucleases into cells has proven challenging as the large size and highly repetitive nature of the TAL effector DNA-binding domain precludes their incorporation into many types of viral vectors. Furthermore, viral and non-viral gene delivery methods carry the risk of insertional mutagenesis and have been shown to increase the off-target activity of site-specific nucleases. We previously demonstrated that direct delivery of zinc-finger nuclease proteins enables highly efficient gene knockout in a variety of mammalian cell types with reduced off-target effects. Here we show that conjugation of cell-penetrating poly-Arg peptides to a surface-exposed Cys residue present on each TAL effector repeat imparted cell-penetrating activity to purified TALEN proteins. These modifications are reversible under reducing conditions and enabled TALEN-mediated gene knockout of the human CCR5 and BMPR1A genes at rates comparable to those achieved with transient transfection of TALEN expression vectors. These findings demonstrate that direct protein delivery, facilitated by conjugation of chemical functionalities onto the TALEN protein surface, is a promising alternative to current non-viral and viral-based methods for TALEN delivery into mammalian cells.

Enhanced Efficiency of Human Pluripotent Stem Cell Genome Editing through Replacing TALENs with CRISPRs

Human pluripotent stem cells (hPSCs) offer unprecedented opportunities to study cellular differentiation and model human diseases. The ability to precisely modify any genomic sequence holds the key to realizing the full potential of hPSCs. Thanks to the rapid development of novel genome editing technologies driven by the enormous interest in the hPSC field, genome editing in hPSCs has evolved from being a daunting task a few years ago to a routine procedure in most laboratories. Here, we provide an overview of the mainstream genome editing tools, including zinc finger nucleases, transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeat/CAS9 RNA-guided nucleases, and helper-dependent adenoviral vectors. We discuss the features and limitations of these technologies, as well as how these factors influence the utility of these tools in basic research and therapies.

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

Targeted gene disruption in somatic zebrafish cells using engineered TALENs

COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites.

Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency

A CRISPR Approach to Gene Targeting

CRISPR Genome Editing: Into the Second Decade

Molecular scissors, such as meganucleases, zinc-finger nucleases (ZFN), and transcription activator-like effector nucleases (TALEN), are valuable tools for generating double-strand breaks (DSB) in the genome that can lead to a functional knockout of the targeted gene or used to integrate a DNA sequence at a specific locus in the genome. Especially in farm animal species from which true pluripotent embryonic stem cells have not been established, these molecular scissors are a new option for engineering the genome in a way that was not feasible before. Meganucleases (also called homing nucleases) are natural proteins found in many single-cell organisms that are mainly involved in the cell’s repair mechanism after a strand break occurs. They are capable of recognising their binding site by identifying a sequence containing between 12 and >30 base pairs. The prototype enzyme for demonstrating DSB stimulation of gene targeting was I-SceI, which has a long recognition site (I-SceI 18 bp). The recognition specificity of enzymes such as I-SceI can be modified to be specific for a desired sequence within the genome. The use of meganucleases to genetically modify organisms has proved very successful in several species, including frog, fly, fish, plants, and human cells, but the intimate connection between the recognition and cleavage elements in the protein structure makes it difficult to alter one without affecting the other. The class of targeting reagents that has proved the most versatile and effective in recent years is that of ZFN. The ZFN possess separate DNA-binding and cleavage domains, which facilitate design according to the desired target. These molecules originate from the natural type IIS restriction enzyme FokI (Li et al. 1992 Proc. Natl. Acad. Sci. USA 89, 4275–4279). The cleavage domain has no sequence specificity and the binding domain can be used to make ZFN specific to a targeted sequence. The requirement for dimerisation of the FokI makes ZFN even more specific and avoids off-target events, as a monomeric cleavage does not occur at single binding sites. One zinc-finger molecule is specific for a base triplet; joining several zinc-finger molecules is sufficient to pick out a single target in a complex genome. ZFN have been used to modify the genome of several species as Xenopus, drosophila, C. elegans, zebrafish, rat, mouse, human cells, hamster cells, rabbit, pigs, and cattle. Different methods have been used to alter the host genomes either by ZFN mRNA or DNA injection into zygotes or by transfection of somatic cells followed by somatic cell nuclear transfer. Even a direct delivery of ZFN proteins can generate a targeted mutation (Gaj et al. 2012 Nat. Methods 9, 805–807). The efficiency of ZFN-mediated knockout was increased up to 10,000-fold compared with traditional gene knockout by homologous recombination. Rarely, off-target events were described but most were located in an intergenic or intronic region of the genome. Transcription activator-like effectors are a family of virulence factors produced by a genus of plant pathogens, Xanthomonas spp. The proteins naturally comprise 17 to 18 repeats of 34 amino acids. The binding specificity is determined by the amino acids at positions 12 and 13 within each repeat. Combined with an endonuclease, TALEs (referred to as TALENs) can be used to specifically target almost any known genomic sequence. The main difference between ZFNs and TALENs is the recognition of the DNA sequence. While ZFNs recognise nucleotide triplets, TALENs recognise single nucleotides, rendering TALENs, in theory, adjustable to any given sequence in a genome while ZFNs need defined prerequisites to be specific. TALENs have already been used to alter the genomes of rats, zebrafish, human iPSCs, and pigs (personal communication). Molecular scissors open a wide range of new applications for modifying the genome of different species or cells with which it has remained very difficult to work. Breeding for agricultural purposes and biomedicine, including the development of large animal models for human diseases and xenotransplantation, will greatly benefit from these new tools. With the advent of ZFN- and TALEN-mediated gene knockouts, mammalian transgenesis has taken a major leap forward as a straightforward technology for gene knockout and knock-in.

Although noncancerous immortalized cell lines have been developed by introducing genes into human and murine somatic cells, such cell lines have not been available in large domesticated animals like pigs. For immortalizing porcine cells, primary porcine fetal fibroblasts were isolated and cultured using the human telomerase reverse transcriptase (hTERT) gene. After selecting cells with neomycin for 2 weeks, outgrowing colonized cells were picked up and subcultured for expansion. Immortalized cells were cultured for more than 9 months without changing their doubling time (~24 hours) or their diameter (< 20 µm) while control cells became replicatively senescent during the same period. Even a single cell expanded to confluence in 100 mm dishes. Furthermore, to knockout the CMAH gene, designed plasmids encoding a transcription activator-like effector nuclease (TALENs) pairs were transfected into the immortalized cells. Each single colony was analyzed by the mutation-sensitive T7 endonuclease I assay, fluorescent PCR, and dideoxy sequencing to obtain three independent clonal populations of cells that contained biallelic modifications. One CMAH knockout clone was chosen and used for somatic cell nuclear transfer. Cloned embryos developed to the blastocyst stage. In conclusion, we demonstrated that immortalized porcine fibroblasts were successfully established using the human hTERT gene, and the TALENs enabled biallelic gene disruptions in these immortalized cells.