ZFN

Agricultural production relies on horticultural crops, including vegetables, fruits, and ornamental plants, which sustain human life. With an alarming increase in human population and the consequential need for more food, it has become necessary for increased production to maintain food security. Conventional breeding has subsidized the development of improved verities but to enhance crop production, new breeding techniques need to be acquired. CRISPR-Cas9 system is a unique and powerful genome manipulation tool that can change the DNA in a precise way...

BACKGROUND: α1-antitrypsin deficiency is most commonly caused by a mutation in exon-7 of SERPINA1 (SA1-ATZ), resulting in hepatocellular accumulation of a misfolded variant (ATZ). Human SA1-ATZ-transgenic (PiZ) mice exhibit hepatocellular ATZ accumulation and liver fibrosis. We hypothesized that disrupting the SA1-ATZ transgene in PiZ mice by in vivo genome editing would confer a proliferative advantage to the genome-edited hepatocytes, enabling them to repopulate the liver. METHODS: To create a targeted DNA break in exon-7 of the SA1-ATZ transgene, we generated 2 recombinant adeno-associated viruses (rAAV) expressing a zinc-finger nuclease pair (rAAV-ZFN), and another rAAV for gene correction by targeted insertion (rAAV-TI)...

Silkworm is a lepidopteran insect that has been used as a model for a wide variety of biological studies. The microinjection technique is available, and it is possible to cause transgenesis as well as target gene disruption via the genome editing technique. TALEN-mediated knockout is especially effective in this species. We also succeeded in the precise and efficient integration of a donor vector using the precise integration into target chromosome (PITCh) method. Here we describe protocols for ZFN (zinc finger nuclease)-, TALEN (transcription activator-like effector nuclease)-, and CRISPR/Cas9-mediated genome editing as well as the PITCh technique in the silkworm...

The development of ZFN, TALEN, and CRISPR/Cas9 systems has simplified the process of generating knockout (KO) and knock-in (KI) rats in addition to mice. However, in rats, an efficient genome editing technique that uses in vitro fertilized oocytes has not been established. Recently, we reported the stable generation of offspring from five standard strains of rats by superovulation and in vitro fertilization (IVF). Furthermore, genome-edited rats can be easily generated by electroporation. First, juvenile female rats are administered LHRH (luteinizing hormone-releasing hormone) to synchronize the estrous cycle and then AIS (Automatic Identification System) with PMSG (pregnant mare serum gonadotropin) before hCG (human chorionic gonadotropin) for superovulation...

Many genetically engineered rat strains have been produced by the development of genome editing technology, although it used to be technical difficulty and low production efficiency. Knockout and knock-in strains can be simple and quick produced using zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Presently, genome edited strains have been produced by microinjection and a new electroporation method named technique for animal knockout system by electroporation (TAKE)...

Many genome-edited mouse and rat strains have been produced using engineered endonucleases, including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Especially, CRISPR-Cas9 is powerful tool that can be easy, rapid, and high-efficiency-produced new genome-edited strains. Furthermore, new technique, Technique for Animal Knockout system by Electroporation (TAKE), efficiently accelerate production of new strains by direct nuclease introduction into intact embryos using electroporation...

Zinc finger nucleases (ZFNs) are programmable nucleases that have contributed significantly to past genome-editing research. They are now utilized much less owing to the advent of transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein system (CRISPR-Cas). These new methods allow for easier generation of reagents that target genomic sequences of interest and are less labor-intensive than ZFNs at targeting desired sequences...

For several decades, genome engineering has raised interest among many researchers and physicians in the study of genetic disorders and their treatments. Compared to its predecessors, zinc-finger nucleases (ZFN) and transcription activator-like effectors (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) is currently the most efficient molecular tool for genome editing. This system, originally identified as a bacterial adaptive immune system, is capable of cutting and modifying any gene of a large number of living organisms...

Stem cells, which can self-renew and differentiate into different cell types, have become the keystone of regenerative medicine due to these properties. With the achievement of superior clinical results in the therapeutic approaches of different diseases, the applications of these cells in the treatment of genetic diseases have also come to the fore. Foremost, conventional approaches of stem cells to genetic diseases are the first approaches in this manner, and they have brought safety issues due to immune reactions caused by allogeneic transplantation...

In October 2020, the chemistry Nobel Prize was awarded to Emmanuelle Charpentier and Jennifer A. Doudna for the discovery of a new promising genome-editing tool: the genetic scissors of CRISPR-Cas9. The identification of CRISPR arrays and the subsequent identification of cas genes, which together represent an adaptive immunological system that exists not only in bacteria but also in archaea, led to the development of diverse strategies used for precise DNA editing, providing new insights in basic research and in clinical practice...

The spread of HIV is on the rise and has become a global issue, especially for underdeveloped and developing countries. This is due to the fact that HIV majorly occurs asymptomatically and is implausible for early diagnosis. Recent advances in research and science have enabled the investigation of a new potential treatment involving gene-based therapy, known as RNA interference (RNAi) that will direct gene silencing and further compensate for natural variants and viral mutants. Several types of small regulatory RNA are discussed in this present study, including microRNA (miRNA), small interfering RNA (siRNA), and short hairpin RNA (shRNA)...

Leucine-rich repeat kinase 2 ( LRRK2 ) gene mutation is an autosomal dominant mutation associated with Parkinson's disease (PD). Among LRRK2 gene mutations, the LRRK2 G2019S mutation is frequently involved in PD onset. Currently, diverse gene correction tools such as zinc finger nucleases (ZFNs), helper-dependent adenoviral vector (HDAdV), the bacterial artificial chromosome-based homologous recombination (BAC-based HR) system, and CRISPR/Cas9-homology-directed repair (HDR) or adenine base editor (ABE) are used in genome editing...

Gene editing with programmable nucleases opens new perspectives in important practice areas, such as healthcare and agriculture. The most challenging problem for the safe and effective therapeutic use of gene editing technologies is the proper delivery and expression of gene editors in cells and tissues of different organisms. Virus-based and nonviral systems can be used for the successful delivery of gene editors. Here we have reviewed structural elements of nonviral DNA- and RNA-based expression vectors for gene editing and delivery methods in vitro and in vivo...

Gene editing with programmable nucleases opens new perspectives in important practice areas, such as healthcare and agriculture. The most challenging problem for the safe and effective therapeutic use of gene editing technologies is the proper delivery and expression of gene editors in cells and tissues of different organisms. Virus-based and nonviral systems can be used for the successful delivery of gene editors. Here we have reviewed structural elements of nonviral DNA- and RNA-based expression vectors for gene editing and delivery methods in vitro and in vivo...

Gene editing techniques have made a significant contribution to the development of better crops. Gene editing enables precise changes in the genome of crops, which can introduce new possibilities for altering the crops' traits. Since the last three decades, various gene editing techniques such as meganucleases, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspersed short palindromic repeats (CRISPR)/Cas (CRISPR-associated proteins) have been discovered...

Zinc finger nuclease (ZFN)-based in vivo genome editing is a novel treatment that can potentially provide lifelong protein replacement with single intravenous administration. Three first-in-human open-label ascending single-dose phase 1/2 studies were performed in parallel (starting November 2017) primarily to assess safety and tolerability of ZFN in vivo editing therapy in mucopolysaccharidosis (MPS) I (n=3), MPS II (n=9), and hemophilia B (n=1). Treatment was well tolerated with no serious treatment-related adverse events...

Cardiovascular diseases (CVDs) are the leading cause of mortality worldwide. However, the lack of human cardiomyocytes with proper genetic backgrounds limits the study of disease mechanisms. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have significantly advanced the study of these conditions. Moreover, hPSC-CMs made it easy to study CVDs using genome-editing techniques. This article discusses the applications of these techniques in hPSC for studying CVDs. Recently, several genome-editing systems have been used to modify hPSCs, including zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9)...

Epigenetic changes are the heritable modifications in genes without altering DNA sequences. The epigenetic changes occur in the plant genomes to regulate gene expression patterns, which were used to regulate different biological processes, including coping various environmental stresses. These changes, including DNA methylation, non-coding RNA regulation, and histone modification, play a vital role in the transcription and translation processes to regulate gene expression. Gene engineering for the development of stress-tolerant crops via the DNA methylation pathway initially needs a proper selection of genes and its promoter...

Genomic mutations are the driving force of biological diversity but they are also the cause of a plethora of human diseases ranging from heritable disorders to neurological pathologies and cancer. For most genetic disorders, there is no curative treatment available to date. The demand for precise, preferably patient-specific, treatment regimen offering cure is naturally high. Genome editing by Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas enables targeted manipulation of genomes, thereby offering the opportunity to treat such diseases...

Climate change gives rise to numerous environmental stresses, including soil salinity. Salinity/salt stress is the second biggest abiotic factor affecting agricultural productivity worldwide by damaging numerous physiological, biochemical, and molecular processes. In particular, salinity affects plant growth, development, and productivity. Salinity responses include modulation of ion homeostasis, antioxidant defense system induction, and biosynthesis of numerous phytohormones and osmoprotectants to protect plants from osmotic stress by decreasing ion toxicity and augmented reactive oxygen species scavenging...