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We present a TALEN-based workflow to generate and maintain dual-edited (IL-15+/+/TGFβR2-/-) iPSCs that produce enhanced iPSC-derived natural killer (iNK) cells for cancer immunotherapy. It involves using a cell lineage promoter for knocking in (KI) gene(s) to minimize the potential effects of expression of any exogenous genes on iPSCs. As a proof-of-principle, we KI IL-15 under the endogenous B2M promoter and show that it results in high expression of the sIL-15 in iNK cells but minimal expression in iPSCs. Furthermore, given that it is known that knockout (KO) of TGFβR2 in immune cells can enhance resistance to the suppressive TGF-β signaling in the tumor microenvironment, we develop a customized medium containing Nodal that can maintain the pluripotency of iPSCs with TGFβR2 KO, enabling banking of these iPSC clones. Ultimately, we show that the dual-edited IL-15+/+/TGFβR2-/- iPSCs can be efficiently differentiated into NK cells that show enhanced autonomous growth and are resistant to the suppressive TGF-β signaling.

Mitochondrial diseases are a heterogeneous group of inherited disorders characterized by mitochondrial dysfunction, and these diseases are often severe or even fatal. Mitochondrial diseases are often caused by mitochondrial DNA mutations. Currently, there is no curative treatment for patients with pathogenic mitochondrial DNA mutations. With the rapid development of traditional gene editing technologies, such as zinc finger nucleases and transcription activator-like effector nucleases methods, there has been a search for a mitochondrial gene editing technology that can edit mutated mitochondrial DNA; however, there are still some problems hindering the application of these methods. The discovery of the DddA-derived cytosine base editor has provided hope for mitochondrial gene editing. In this paper, we will review the progress in the research on several mitochondrial gene editing technologies with the hope that this review will be useful for further research on mitochondrial gene editing technologies to optimize the treatment of mitochondrial diseases in the future.

MicroRNAs (miRNAs) are 20-24 nucleotides (nt) small RNAs functioning in eukaryotes. The length and sequence of miRNAs are not only related to the biogenesis of miRNAs but are also important for downstream physiological processes like ta-siRNA production. To investigate these roles, it is informative to create small mutations within mature miRNA sequences. We used both TALENs (transcription activator-like effector nucleases) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to introduce heritable base pair mutations in mature miRNA sequences. For rice, TALEN constructs were built targeting five different mature miRNA sequences and yielding heritable mutations. Among the resulting mutants, mir390 mutant showed a severe defect in the shoot apical meristem (SAM), a shootless phenotype, which could be rescued by the wild-type MIR390. Small RNA sequencing showed the two base pair deletion in mir390 substantially interfered with miR390 biogenesis. In Arabidopsis, CRISPR/Cas9-mediated editing of the miR160* strand confirmed that the asymmetric structure of miRNA is not a necessary determinant for secondary siRNA production. CRISPR/Cas9 with double-guide RNAs successfully generated mir160a null mutants with fragment deletions, at a higher efficiency than a single-guide RNA. The difference between the phenotypic severity of miR160a mutants in Col-0 versus Ler backgrounds highlights a diverged role for miR160a in different ecotypes. Overall, we demonstrated that TALENs and CRISPR/Cas9 are both effective in modifying miRNA precursor structure, disrupting miRNA processing and generating miRNA null mutant plants.

Simple Summary: Follicle-stimulating hormone (fsh) is crucial to the reproduction of fish. In the current investigation, transcription activator-like effector nuclease (TALEN) plasmids targeting the channel catfish fsh gene were electroporated into fertilized eggs to generate infertile channel catfish (Ictalurus punctatus). Human chorionic gonadotropin (HCG) hormone treatment enhanced the spawning and hatching rates of catfish fsh-knockout mutants when lowered infertility was detected. Gene editing of channel catfish offers promise for reproductive confinement of genetically modified, native, and invasive fish to eliminate genetic drift into external ecosystems. Follicle-stimulating hormone (fsh) plays an important role in sexual maturation in catfish. Knocking out the fsh gene in the fish zygote should suppress the reproduction of channel catfish (Ictalurus punctatus). In this study, transcription activator-like effector nuclease (TALEN) plasmids targeting the fsh gene were electroporated into fertilized eggs with the standard double electroporation technique. Targeted fsh cleavage efficiency was 63.2% in P1fsh-knockout catfish. Ten of fifteen (66.7%) control pairs spawned, and their eggs had 32.3–74.3% average hatch rates in 2016 and 2017. Without hormone therapy, the spawning rates of P1 mutants ranged from 33.3 to 40.0%, with an average egg hatching rate of 0.75%. After confirmation of the low fertility of P1 mutants in 2016, human chorionic gonadotropin (HCG) hormone therapy improved the spawning rates by 80% for female mutants and 88.9% for male mutants, and the mean hatch rate was 35.0% for F1 embryos, similar to that of the controls (p > 0.05). Polymerase chain reaction (PCR) identification showed no potential TALEN plasmid integration into the P1 channel catfish genome. Neither the P1 nor the F1 mutant fish showed any noticeable changes in in body weight, survival rate, and hatching rate when the reproductive gene was knocked out. F1 families had a mean inheritance rate of 50.3%. The results brought us one step closer to allowing implementation of certain genetic techniques to aquaculture and fisheries management, while essentially eliminating the potential environment risk posed by transgenic, hybrid, and exotic fish as well as domestic fish. [ABSTRACT FROM AUTHOR]

X-linked hyper-immunoglobulin M (hyper-IgM) syndrome (XHIM) is a primary immunodeficiency due to mutations in CD40 ligand that affect immunoglobulin class-switch recombination and somatic hypermutation. The disease is amenable to gene therapy using retroviral vectors, but dysregulated gene expression results in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) platforms to efficiently drive integration of a normal copy of the CD40L cDNA delivered by Adeno-Associated Virus. Site-specific insertion of the donor sequence downstream of the endogenous CD40L promoter maintained physiologic expression of CD40L while overriding all reported downstream mutations. High levels of gene modification were achieved in primary human hematopoietic stem cells (HSCs), as well as in cell lines and XHIM-patient-derived T cells. Notably, gene-corrected HSCs engrafted in immunodeficient mice at clinically relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM.

Adoptive cell therapy using chimeric antigen receptor (CAR) T cells has proven to be lifesaving for many cancer patients. However, its therapeutic efficacy has been limited in solid tumors. One key factor for this is cancer-associated fibroblasts (CAFs) that modulate the tumor microenvironment (TME) to inhibit T cell infiltration and induce "T cell dysfunction." Additionally, the sparsity of tumor-specific antigens (TSA) and expression of CAR-directed tumor-associated antigens (TAA) on normal tissues often results in "on-target off-tumor" cytotoxicity, raising safety concerns. Using TALEN-mediated gene editing, we present here an innovative CAR T cell engineering strategy to overcome these challenges. Our allogeneic "Smart CAR T cells" are designed to express a constitutive CAR, targeting FAP + CAFs in solid tumors. Additionally, a second CAR targeting a TAA such as mesothelin is specifically integrated at a TCR signaling-inducible locus like PDCD1. FAPCAR-mediated CAF targeting induces expression of the mesothelin CAR, establishing an IF/THEN-gated circuit sensitive to dual antigen sensing. Using this approach, we observe enhanced anti-tumor cytotoxicity, while limiting "on-target off-tumor" toxicity. Our study thus demonstrates TALEN-mediated gene editing capabilities for design of allogeneic IF/THEN-gated dual CAR T cells that efficiently target immunotherapy-recalcitrant solid tumors while mitigating potential safety risks, encouraging clinical development of this strategy., Competing Interests: Declaration of interests S.Dharani, H.C., A.J., J.V., P.D., L.P., and S.Das are current employees and equity holders at Cellectis. TALEN is a Cellectis patented technology. J.P.F. is a former Cellectis employee and is a current employee of Bristol Myers Squibb., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Microbial production of chemicals and proteins from biomass-derived and waste sugar streams is a rapidly growing area of research and development. While the model yeast Saccharomyces cerevisiae is an excellent host for the conversion of glucose to ethanol, production of other chemicals from alternative substrates often requires extensive strain engineering. To avoid complex and intensive engineering of S. cerevisiae, other yeasts are often selected as hosts for bioprocessing based on their natural capacity to produce a desired product: for example, the efficient production and secretion of proteins, lipids, and primary metabolites that have value as commodity chemicals. Even when using yeasts with beneficial native phenotypes, metabolic engineering to increase yield, titer, and production rate is essential. The non-conventional yeasts Kluyveromyces lactis, K. marxianus, Scheffersomyces stipitis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris have been developed as eukaryotic hosts because of their desirable phenotypes, including thermotolerance, assimilation of diverse carbon sources, and high protein secretion. However, advanced metabolic engineering in these yeasts has been limited. This review outlines the challenges of using non-conventional yeasts for strain and pathway engineering, and discusses the developed solutions to these problems and the resulting applications in industrial biotechnology.

The β2-adrenergic receptor (β2AR) has provided a paradigm to elucidate how G protein-coupled receptors (GPCRs) control intracellular signaling, including the discovery that β-arrestins, which bind to ligand-activated GPCRs, are central for GPCR function. We used genome editing, conditional gene deletion, and small interfering RNAs (siRNAs) to determine the roles of β-arrestin 1 (β-arr1) and β-arr2 in β2AR internalization, trafficking, and signaling to ERK. We found that only β-arr2 was essential for β2AR internalization. Unexpectedly, β-arr1 and β-arr2 and receptor internalization were dispensable for ERK activation. Instead, β2AR signaled through Gαs and Gβγ subunits through a pathway that involved the tyrosine kinase SRC, the adaptor protein SHC, the guanine nucleotide exchange factor SOS, the small GTPase RAS, and the kinases RAF and MEK, which led to ERK activation. These findings provide a molecular framework for β2AR signaling through β-arrestin-independent pathways in key physiological functions and under pathological conditions.

The amphibian model Xenopus, has been used extensively over the past century to study multiple aspects of cell and developmental biology. Xenopus offers advantages of a non-mammalian system, including high fecundity, external development, and simple housing requirements, with additional advantages of large embryos, highly conserved developmental processes, and close evolutionary relationship to higher vertebrates. There are two main species of Xenopus used in biomedical research, Xenopus laevis and Xenopus tropicalis; the common perception is that both species are excellent models for embryological and cell biological studies, but only Xenopus tropicalis is useful as a genetic model. The recent completion of the Xenopus laevis genome sequence combined with implementation of genome editing tools, such as TALENs (transcription activator-like effector nucleases) and CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated nucleases), greatly facilitates the use of both Xenopus laevis and Xenopus tropicalis for understanding gene function in development and disease. In this paper, we review recent advances made in Xenopus laevis and Xenopus tropicalis with TALENs and CRISPR-Cas and discuss the various approaches that have been used to generate knockout and knock-in animals in both species. These advances show that both Xenopus species are useful for genetic approaches and in particular counters the notion that Xenopus laevis is not amenable to genetic manipulations.

Thalassemias are monogenic hematologic diseases that are classified as α- or β-thalassemia according to its quantitative abnormalities of adult α- or β-globin chains. β-thalassemia has widely spread throughout the world especially in Mediterranean countries, the Middle East, Central Asia, India, Southern China, and the Far East as well as countries along the north coast of Africa and in South America. The one and the only cure for β-thalassemia is allogenic hematopoietic stem cell transplantations (HSCT). Nevertheless, the difficulty to find matched donors has hindered the availability of this therapeutic option. Therefore, this present review explored the alternatives for β-thalassemia treatment such as RNA manipulation therapy, splice-switching, genome editing and generation of corrected induced pluripotent stem cells (iPSCs). Manipulation of β-globin RNA is mediated by antisense oligonucleotides (ASOs) or splice-switching oligonucleotides (SSOs), which redirect pre-mRNA splicing to significantly restore correct β-globin pre-mRNA splicing and gene product in cultured erythropoietic cells. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) are designer proteins that can alter the genome precisely by creating specific DNA double-strand breaks. The treatment of β-thalassemia patient-derived iPSCs with TALENs have been found to correct the β-globin gene mutations, implying that TALENs could be used as a therapy option for β-thalassemia. Additionally, CRISPR technologies using Cas9 have been used to fix mutations in the β-globin gene in cultured cells as well as induction of hereditary persistence of fetal hemoglobin (HPFH), and α-globin gene deletions have proposed a possible therapeutic option for β-thalassemia. Overall, the accumulated research evidence demonstrated the potential of ASOs-mediated aberrant splicing correction of β-thalassemia mutations and the advancements of genome therapy approaches using ZFNs, TALENs, and CRISPR/Cas9 that provided insights in finding the permanent cure of β-thalassemia.

Follicle-stimulating hormone (fsh) plays an important role in sexual maturation in catfish. Knocking out the fsh gene in the fish zygote should suppress the reproduction of channel catfish (Ictalurus punctatus). In this study, transcription activator-like effector nuclease (TALEN) plasmids targeting the fsh gene were electroporated into fertilized eggs with the standard double electroporation technique. Targeted fsh cleavage efficiency was 63.2% in P1fsh-knockout catfish. Ten of fifteen (66.7%) control pairs spawned, and their eggs had 32.3–74.3% average hatch rates in 2016 and 2017. Without hormone therapy, the spawning rates of P1 mutants ranged from 33.3 to 40.0%, with an average egg hatching rate of 0.75%. After confirmation of the low fertility of P1 mutants in 2016, human chorionic gonadotropin (HCG) hormone therapy improved the spawning rates by 80% for female mutants and 88.9% for male mutants, and the mean hatch rate was 35.0% for F1 embryos, similar to that of the controls (p > 0.05). Polymerase chain reaction (PCR) identification showed no potential TALEN plasmid integration into the P1 channel catfish genome. Neither the P1 nor the F1 mutant fish showed any noticeable changes in in body weight, survival rate, and hatching rate when the reproductive gene was knocked out. F1 families had a mean inheritance rate of 50.3%. The results brought us one step closer to allowing implementation of certain genetic techniques to aquaculture and fisheries management, while essentially eliminating the potential environment risk posed by transgenic, hybrid, and exotic fish as well as domestic fish.

Simple Summary: Gonadotropin-releasing hormone (GnRH) plays a pivotal role in fish reproduction. In the present study, transcription activator-like effector nuclease (TALEN) plasmids targeting the catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). After low fertility was observed, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants (individuals with human-induced sequence changes at the cfGnRH locus). Gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising. Transcription activator-like effector nuclease (TALEN) plasmids targeting the channel catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). Targeted cfGnRH fish were sequenced and base deletion, substitution, and insertion were detected. The gene mutagenesis was achieved in 52.9% of P1 fish. P1 mutants (individuals with human-induced sequence changes at the cfGnRH locus) had lower spawning rates (20.0–50.0%) when there was no hormone therapy compared to the control pairs (66.7%) as well as having lower average egg hatch rates (2.0% versus 32.3–74.3%) except for one cfGnRH mutated female that had a 66.0% hatch rate. After low fertility was observed in 2016, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants in 2017, which were not significantly different from the controls (p > 0.05). No exogenous DNA fragments were detected in the genome of mutant P1 fish, indicating no integration of the plasmids. No obvious effects on other economically important traits were observed after the knockout of the reproductive gene in the P1 fish. Growth rates, survival, and appearance between mutant and control individuals were not different. While complete knock-out of reproductive output was not achieved, as these were mosaic P1 brood stock, gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising. [ABSTRACT FROM AUTHOR]

Targeted genome editing technology can correct the sickle cell disease mutation of the β-globin gene in hematopoietic stem cells. This correction supports production of red blood cells that synthesize normal hemoglobin proteins. Here, we demonstrate that Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system can target DNA sequences around the sickle-cell mutation in the β-globin gene for site-specific cleavage and facilitate precise correction when a homologous donor template is codelivered. Several pairs of TALENs and multiple CRISPR guide RNAs were evaluated for both on-target and off-target cleavage rates. Delivery of the CRISPR/Cas9 components to CD34+ cells led to over 18% gene modification in vitro. Additionally, we demonstrate the correction of the sickle cell disease mutation in bone marrow derived CD34+ hematopoietic stem and progenitor cells from sickle cell disease patients, leading to the production of wild-type hemoglobin. These results demonstrate correction of the sickle mutation in patient-derived CD34+ cells using CRISPR/Cas9 technology.

Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR-based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator-like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology.

Abstract Methodologies of genome editing are rapidly developing with the improvement of gene science and technology, mechanism‐based understanding, and urgent needs. In addition to the specificity and efficiency of on‐target sites, one of the most important issues is to find and avoid off‐targets before clinical application of gene editing as a therapy. Various algorithms, modified nucleases, and delivery vectors are developed to localize and minimize off‐target sites. The present review aimed to clarify off‐targets of various genome editing and explore potentials of clinical application by understanding structures, mechanisms, clinical applications, and off‐target activities of genome editing systems, including CRISPR/Cas9, CRISPR/Cas12a, zinc finger nucleases, transcription activator‐like effector nucleases, meganucleases, and recent developments. Current genome editing in cancer therapy mainly targeted immune systems in tumor microenvironment by ex vivo modification of the immune cells in phases I/II of clinical trials. We believe that genome editing will be the critical part of clinical precision medicine strategy and multidisciplinary therapy strategy by integrating gene sequencing, clinical transomics, and single cell biomedicine. There is an urgent need to develop on/off‐target‐specific biomarkers to monitor the efficacy and side‐effects of gene therapy. Thus, the genome editing will be an alternative of clinical therapies for cancer with the rapid development of methodology and an important part of clinical precision medicine strategy.

Transposon-based genetic transformation has facilitated insect functional genomics and new strategies of pest management. However, there is a need for alternative, site-specific approaches to overcome limitations of random integration (and associated position-effects) and potential instability of inserted transgenes. Here we describe a transposon-free, site-specific genetic transformation system mediated by transcription activator-like effector nucleases (TALENs) in the silkworm, Bombyx mori, a lepidopteran model insect. We successfully established a site-specific transgenic system with comparable transformation efficiency to transposon-based genetic transformation through microinjection of TALENs mRNA targeting the BmBLOS2 locus and a linearizable donor plasmid encoding an expression cassette of the DsRed2 red fluorescent protein. This system provides a valuable approach for insect transgenesis and will enable future functional gene analysis and generate novel applications in agricultural and medical insect pest-management technologies.

CRISPR/Cas9 technology is a highly promising genome editing tool in the mouse, potentially overcoming the costs and time required for more traditional gene targeting methods in embryonic stem (ES) cells. Recently, compared to the wildtype nuclease, paired Cas9 nickase (Cas9n) combined with single guide RNA (sgRNA) molecules has been found to enhance the specificity of genome editing while reducing off-target effects. Paired Cas9n has been shown to be as efficient as Cas9 for generating insertion and deletion (indel) mutations by non-homologous end joining and targeted deletion in the genome. However, an efficient and reliable approach to the insertion of loxP sites flanking critical exon(s) to create a conditional allele of a target gene remains an elusive goal. In this study, we microinjected Cas9n RNA with sgRNAs together with a single DNA template encoding two loxP sites flanking (floxing) exon 2 of the isoprenoid synthase containing domain (Ispd) into the pronucleus and cytoplasm of C57BL/6NCr one-cell stage zygotes. After surgical transfer, one F0 mouse expressing a conditional allele was produced (at a frequency of ∼8% of live pups born). The floxed allele was transmitted through the germline to F1 progeny, and could be successfully recombined using Cre recombinase. This study indicates that conditional targeting can be accomplished effectively using paired Cas9n and a single DNA template.

Transcription activator-like effector nuclease (TALEN) plasmids targeting the channel catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). Targeted cfGnRH fish were sequenced and base deletion, substitution, and insertion were detected. The gene mutagenesis was achieved in 52.9% of P1 fish. P1 mutants (individuals with human-induced sequence changes at the cfGnRH locus) had lower spawning rates (20.0–50.0%) when there was no hormone therapy compared to the control pairs (66.7%) as well as having lower average egg hatch rates (2.0% versus 32.3–74.3%) except for one cfGnRH mutated female that had a 66.0% hatch rate. After low fertility was observed in 2016, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants in 2017, which were not significantly different from the controls (p > 0.05). No exogenous DNA fragments were detected in the genome of mutant P1 fish, indicating no integration of the plasmids. No obvious effects on other economically important traits were observed after the knockout of the reproductive gene in the P1 fish. Growth rates, survival, and appearance between mutant and control individuals were not different. While complete knock-out of reproductive output was not achieved, as these were mosaic P1 brood stock, gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising.

Cytoplasmic male sterility (CMS), encoded by the mitochondrial open reading frames (ORFs), has long been used to economically produce crop hybrids. However, the utilization of CMS also hinders the exploitation of sterility and fertility variation in the absence of a restorer line, which in turn narrows the genetic background and reduces biodiversity. Here, we used a mitochondrial targeted transcription activator-like effector nuclease (mitoTALENs) to knock out ORF138 from the Ogura CMS broccoli hybrid. The knockout was confirmed by the amplification and re-sequencing read mapping to the mitochondrial genome. As a result, knockout of ORF138 restored the fertility of the CMS hybrid, and simultaneously manifested a cold-sensitive male sterility. ORF138 depletion is stably inherited to the next generation, allowing for direct use in the breeding process. In addition, we proposed a highly reliable and cost-effective toolkit to accelerate the life cycle of fertile lines from CMS-derived broccoli hybrids. By applying the k-mean clustering and interaction network analysis, we identified the central gene networks involved in the fertility restoration and cold-sensitive male sterility. Our study enables mitochondrial genome editing via mitoTALENs in Brassicaceae vegetable crops and provides evidence that the sex production machinery and its temperature-responsive ability are regulated by the mitochondria., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Methodologies of genome editing are rapidly developing with the improvement of gene science and technology, mechanism‐based understanding, and urgent needs. In addition to the specificity and efficiency of on‐target sites, one of the most important issues is to find and avoid off‐targets before clinical application of gene editing as a therapy. Various algorithms, modified nucleases, and delivery vectors are developed to localize and minimize off‐target sites. The present review aimed to clarify off‐targets of various genome editing and explore potentials of clinical application by understanding structures, mechanisms, clinical applications, and off‐target activities of genome editing systems, including CRISPR/Cas9, CRISPR/Cas12a, zinc finger nucleases, transcription activator‐like effector nucleases, meganucleases, and recent developments. Current genome editing in cancer therapy mainly targeted immune systems in tumor microenvironment by ex vivo modification of the immune cells in phases I/II of clinical trials. We believe that genome editing will be the critical part of clinical precision medicine strategy and multidisciplinary therapy strategy by integrating gene sequencing, clinical transomics, and single cell biomedicine. There is an urgent need to develop on/off‐target‐specific biomarkers to monitor the efficacy and side‐effects of gene therapy. Thus, the genome editing will be an alternative of clinical therapies for cancer with the rapid development of methodology and an important part of clinical precision medicine strategy. [ABSTRACT FROM AUTHOR]

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Plant genetic engineering is one of the most significant tools implemented in the modern molecular crop breeding techniques. The conventional approaches of plant genetic transformation include Agrobacterium tumefaciens, particle bombardment, DNA uptake into protoplast. The transgenic events derived by these methods carry the transgenes that are integrated at random sites in the plant genome. Novel techniques that mediate integration of foreign genes at specific pre-determined locations circumvent many problems associated with the existing methods of gene transfer. The recent years have witnessed the emergence of gene targeting techniques by employing zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindrome repeats (CRISPR). The present review focuses on the various approaches and their performance of plant gene targeting and suggests future directions in the important areas of plant molecular biology.

The ability to precisely change the deoxyribonucleic acid (DNA) bases at specific sites offers tremendous advantages in the field of molecular biology and medical biotechnology. Identification of Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR), revelation of its role in prokaryotic adaptive immunity and subsequent conversion into genome and epigenome engineering system are the landmark research progresses of the decade. The possibilities of deciphering the molecular mechanisms of the disease, identifying the disease targets, generating the disease models, validating the drug targets, developing resistance to the infection and correcting the genotype have brought off much enthusiasm in the field of infectious diseases and genetic disorders. This review focuses on CRISPR/Cas9's impact in the field of infection and genetic disorders. [ABSTRACT FROM AUTHOR]

Objective To create genetically modified goat as a biopharming source of recombinant human lacotoferrin (hLF) with transcription activator-like effector nucleases. Methods TALENs and targeting vector were transferred into cultured fibroblasts to insert hLF cDNA in the goat beta-lactoglobulin (BLG) locus with homology-directed repair. The gene targeted efficiency was checked using sequencing and TE7I assay. The bi-allelic gene targeted colonies were isolated and confirmed with polymerase chain reaction, and used as donor cells for somatic cell nuclear transfer (SCNT). Results The targeted efficiency for BLG gene was approximately 10%. Among 12 Bi-allelic gene targeted colonies, five were used in first round SCNT and 4 recipients (23%) were confirmed pregnant at 30 d. In second round SCNT, 7 (53%), 4 (31%), and 3 (23%) recipients were confirmed to be pregnant by ultrasound on 30 d, 60 d, and 90 d. Conclusion This finding signifies the combined use of TALENs and SCNT can generate bi-allelic knock-in fibroblasts that can be cloned in a fetus. Therefore, it might lay the foundation for transgenic hLF goat generation and possible use of their mammary gland as a bioreactor for large-scale production of recombinant hLF.

Background: Echinoderms have long been utilized as experimental materials to study the genetic control of developmental processes and their evolution. Among echinoderms, the molecular study of starfish embryos has received considerable attention across research topics such as gene regulatory network evolution and larval regeneration. Recently, experimental techniques to manipulate gene functions have been gradually established in starfish as the feasibility of genome editing methods was reported. However, it is still unclear when these techniques cause genome cleavage during the development of starfish, which is critical to understand the timeframe and applicability of the experiment during early development of starfish., Results: We herein reported that gene functions can be analyzed by the genome editing method TALEN in early embryos, such as the blastula of the starfish Patiria pectinifera. We injected the mRNA of TALEN targeting rar, which was previously constructed, into eggs of P. pectinifera and examined the efficiency of genome cleavage through developmental stages from 6 to 48 hours post fertilization., Conclusion: The results will be key knowledge not only when designing TALEN-based experiments but also when assessing the results., (© 2023 American Association for Anatomy.)

Transcription activator‐like effector nucleases (TALENs) are a novel class of genome editing tools that provide precise insertion, deletion, or substitution of specific genes in order to alter the genome. TALENs consist of a designed TALE domain that mimics the natural transcription activator‐like effector proteins and a nuclease that can cleave DNA in cells. TALENs have recently emerged as a revolutionary genome editing tools in numerous species and cell types. Here, we review the development of TALEN technology and summarize the principles and tools for TALEN‐mediated genome modification in plant cells.

Myostatin (MSTN) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs) in tandem with single-stranded DNA oligonucleotides (ssODNs). We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals.

Well-established targeted technologies to engineer genomes such as zinc-finger nuclease-based editing (ZFN), transcription activator-like effector nuclease-based editing (TALEN), and clustered regularly interspaced short palindromic repeats and associated protein system-based editing (CRISPR/Cas) are proving to advance basic and applied research in numerous plant species. Compared with systems using ZFNs and TALENs, the most recently developed CRISPR/Cas system is more efficient due to its use of an RNA-guided nuclease to generate double-strand DNA breaks. To accelerate the applications of these technologies, we provide here a detailed overview of these systems, highlight the strengths and weaknesses of each, summarize research advances made with these technologies in model and crop plants, and discuss their applications in plant functional genomics. Such targeted approaches for genetically modifying plants will benefit agricultural production in the future. [ABSTRACT FROM AUTHOR]

Reporter embryonic stem cell (ESC) lines with tissue-specific reporter genes may contribute to optimizing the differentiation conditions in vitro as well as trafficking transplanted cells in vivo. To optimize and monitor endothelial cell (EC) differentiation specifically, here we targeted the enhanced green fluorescent protein (EGFP) reporter gene at the junction of 5'UTR and exon2 of the endothelial specific marker gene CD144 using TALENs in human ESCs (H9) to generate a EGFP-CD144-reporter ESC line. The reporter cells expressed EGFP and CD144 increasingly and specifically without unexpected effects during the EC differentiation. The EC differentiation protocol was optimized and applied to EC differentiation from hiPSCs, resulting in an efficient and simplified endothelial differentiation approach. Here we created our own optimized and robust protocol for EC differentiation of hESCs and hiPSCs by generating the lineage- specific site- specific integration reporter cell lines, showing great potential to be applied in the fields such as trafficking gene and cell fate in vivo in preclinical animal models. [ABSTRACT FROM AUTHOR]

Programmable nucleases including zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein have tremendous potential biological and therapeutic applications as novel genome editing tools. These nucleases enable precise modification of the gene of interest by disruption, insertion, or correction. The application of genome editing technology to pluripotent stem cells or hematopoietic stem cells has the potential to remarkably advance the contribution of this technology to life sciences. Specifically, disease models can be generated and effective therapeutics can be developed with great efficiency and speed. Here we review the characteristics and mechanisms of each programmable nuclease. In addition, we review the applications of these nucleases to stem cells for disease therapies and summarize key studies of interest. [ABSTRACT FROM AUTHOR]

Plant genetic engineering is one of the most significant tools implemented in the modern molecular crop breeding techniques. The conventional approaches of plant genetic transformation include Agrobacterium tumefaciens , particle bombardment, DNA uptake into protoplast. The transgenic events derived by these methods carry the transgenes that are integrated at random sites in the plant genome. Novel techniques that mediate integration of foreign genes at specific pre-determined locations circumvent many problems associated with the existing methods of gene transfer. The recent years have witnessed the emergence of gene targeting techniques by employing zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindrome repeats (CRISPR). The present review focuses on the various approaches and their performance of plant gene targeting and suggests future directions in the important areas of plant molecular biology. [ABSTRACT FROM AUTHOR]