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

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

Targeting DNA With Fingers and TALENs.

338. Evaluation of TALENs and the CRISPR/Cas9 Nuclease System To Correct the Sickle Cell Disease Mutation

Ribosomal Proteins Rps19 and Rps14 Cooperate As Tumor Suppressor Genes with p53

103. Pas de Deux – A Single Inducible High-Capacity Adenoviral Vector for Delivery of a Functional TALEN Pair Directed Against Human Dystrophin for the Treatment of Duchenne Muscular Dystrophy (DMD)

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.

562. Modification of TCR Specificity by TALEN and CRISPR

Creation of gene-specific rice mutants by AvrXa23-based TALENs

Gene Editing on Center Stage

At present, the main gene editing tools encompass TREN, Zinc Finger Nucleases (ZFN), clustered regularly interspaced short palindromic repeats (CRISPR), and Transcription Activator-Like Effector Nucleases (TALEN). In this study, we introduce an overview of the three gene editing methodologies and discuss their current clinical applications. In addition, we suggest some trends and future applications within the field of gene editing. ZFNs represent one of the pioneering technologies, demonstrating significant efficacy in mitigating a multitude of genetic diseases and finding applications in agriculture. Yet, this technology contains intricate processes and produces substantial costs when implemented. TALENs have already been employed across various domains. In the medical field, they have been successfully applied in the treatment of leukemia in infants. However, TALENs are being replaced by CRISPR due to the superior efficiency of CRISPR. CRISPR, consisting of six components, exhibits considerable promise in the medical realm, particularly in the context of treating diseases such as Alzheimer's disease (AD). In the realm of genetic engineering, it can collaborate with B cells to rectify specific genes within the human genome, which have been tested in experiments. In the future, it can be used in many fields, including agriculture and nucleic acid testing.

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

Hyperactivation of the Wnt pathway is a common hallmark of many cancers. Deregulation of the pathway is mostly due to mutations in the tumor suppressor gene Adenomatous Polyposis Coli (APC) or the transcriptional regulator of the Wnt pathway β-catenin (CTNNB1). Heterozygous germline mutations in the Mutation Cluster Region (MCR) of the APC gene cause Familial Adenomatous Polyposis (FAP), a condition characterized by multiple colorectal polyps, which over time become malignant. FAP patients also frequently suffer from extracolonic manifestations, such as Congenital Hypertrophy of the Retinal Pigment Epithelium (CHRPE), desmoid tumors and medulloblastoma. Despite many mouse models and the clear etiology of colorectal cancer and FAP, no drugs targeting the Wnt pathway have reached the clinic and there is a need for new genetic animal models. The recent TALEN (Transcription Activator Like Effector Nuclease) and CRISPR/Cas9 gene targeting technologies open the door for novel genetic cancer models. Xenopus tropicalis, an aquatic tetrapod with a true diploid genome, offers unique experimental opportunities to model human cancer. We have adopted the TALEN technology in Xenopus tropicalis to generate a model for Wnt deregulated cancer, by targeting either APC or β-catenin. Targeting of the apc gene in the MCR region induces phenotypes reminiscent of FAP in tadpoles and froglets, including hyperplasia of the intestinal epithelium, desmoid tumors, retinal hyperproliferation and medulloblastoma. These neoplasms show bi-allelic truncating mutations in the apc gene, associated with activation of the Wnt signalling pathway and increased cell proliferation. Secondly, we generated a more direct model for the hyperactivation of the Wnt pathway by developing TALENs against the Ser33 phosphorylation site of β-catenin. Small in frame deletions removing this phosphorylation site will result in a stabilized dominant active form of β-catenin, activating Wnt signalling at the transcriptional endpoint. Injection of this TALEN pair induces similar neoplasms as apc TALEN injection, including desmoid and brain tumors, indicating that these tumors are the consequence of activated Wnt signalling, rather than any of the other disrupted functions of APC. In addition we were able to achieve very efficient double bi-allelic mutation of apc and other genes in different tumors by co-injection of two TALEN pairs. This creates the opportunity to use our model for therapeutic target validation, simply by co-injection of TALENs (or CRISPR/Cas9) against potential targets together with the apc TALENs. We have developed the first genetic cancer models in Xenopus tropicalis by using apc or β-catenin TALENs. These models closely resemble human FAP and will be used for preclinical drug screening and to evaluate potential novel therapeutic targets by multiplexed gene targeting via TALEN and CRISPR/Cas9 technology. Citation Format: Tom Van Nieuwenhuysen, Thomas Naert, David Creytens, Frans Van Roy, Kris Vleminckx. TALEN mediated mutation of apc and β-catenin in Xenopus tropicalis as powerful models for Wnt driven cancer and Familial Adenomatous Polyposis (FAP). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5140. doi:10.1158/1538-7445.AM2015-5140

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.

121. T Cell Receptor Modification by Highly Specific TALEN and CRISPR/Cas9

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.