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Current status of the application of gene editing in pigs.
Genetically modified animals, especially rodents, are widely used in biomedical research. However, non-rodent models are required for efficient translational medicine and preclinical studies. Owing to the similarity in the physiological traits of pigs and humans, genetically modified pigs may be a valuable resource for biomedical research. Somatic cell nuclear transfer (SCNT) using genetically modified somatic cells has been the primary method for the generation of genetically modified pigs. However, site-specific gene modification in porcine cells is inefficient and requires laborious and time-consuming processes. Recent improvements in gene-editing systems, such as zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas) system, represent major advances. The efficient introduction of site-specific modifications into cells via gene editors dramatically reduces the effort and time required to generate genetically modified pigs. Furthermore, gene editors enable direct gene modification during embryogenesis, bypassing the SCNT procedure. The application of gene editors has progressively expanded, and a range of strategies is now available for porcine gene engineering. This review provides an overview of approaches for the generation of genetically modified pigs using gene editors, and highlights the current trends, as well as the limitations, of gene editing in pigs.
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Advancing nutritional quality in oilseed crops through genome editing: a comprehensive review.
Genome editing has emerged as a powerful approach to enhancing the nutritional quality of oilseed crops. Clustered regularly interspaced short palindromic repeats case9 (CRISPR/Cas9) is the predominant editing tool, while transcription activator-like effector nucleases (TALENs) and base editors are used less commonly. Key fatty acid desaturase genes such as FAD2 and FAD3 are prime targets because of their critical functions in fatty acid desaturation. This review summarizes recent progress in editing genes associated with oil composition and related traits across oilseed species. Visual data representations including, Sankey diagrams, heat maps, and crop-trait matrices illustrate shared editing priorities and emerging trait targets across crops. Despite its promise, genome editing still faces challenges in transformation efficiency, field-level validation, and regulatory acceptance. This review underscores the increasing impact of target gene editing on nutritional trait improvement and its potential to accelerate the development of healthier, more sustainable oilseed varieties.
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Genome editing using Cas9 nickases.
The RNA-guided, sequence-specific endonuclease Cas9 has been widely adopted as genome engineering tool due to its efficiency and ease of use. Derived from the microbial CRISPR (clustered regularly interspaced short palindromic repeat) type II adaptive immune system, Cas9 has now been successfully engineered for genome editing applications in a variety of animal and plant species. To reduce potential off-target mutagenesis by wild-type Cas9, homology- and structure-guided mutagenesis of Streptococcus pyogenes Cas9 catalytic domains has produced "nicking" enzymes (Cas9n) capable of inducing single-strand nicks rather than double-strand breaks. Since nicks are generally repaired with high fidelity in eukaryotic cells, Cas9n can be leveraged to mediate highly specific genome editing, either via nonhomologous end-joining or homology-directed repair. Here we describe the preparation, testing, and application of Cas9n reagents for precision mammalian genome engineering.
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Engineered CRISPRa System for Precise and Multiplex Gene Regulation Using a Hybrid dCas12a Variant and Hairpin-Spacer crRNAs.
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a nucleases have emerged as a promising alternative to CRISPR-Cas9 in gene editing and expression regulation. However, the adoption of Cas12a has been hindered due to general off-target activities and limited efficiency. Here, we utilized a hybrid engineered Cas12a variant and hairpin-spacer crRNAs (h-CAP) to enhance the specificity and efficiency of the CRISPR-Cas12a system. Leveraging the h-CAP strategy, we demonstrate both single-base-specific and multiplex gene expression regulation in human cells.
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Clustered Regularly Interspaced Short Palindromic Repeats: Challenges in Treating Retinal Disease.
Ophthalmic researchers and clinicians arguably have led the way for safe, effective gene therapy, most notably with adeno-associated viral gene supplementation in the treatment for patients with Leber congenital amaurosis type 2 with mutations in the RPE65 gene. These successes notwithstanding, most other genetic retinal disease will be refractory to supplementation. The ideal gene therapy approach would correct gene mutations to restore normal function in the affected cells. Gene editing in which a mutant allele is inactivated or converted to sequence that restores normal function is hypothetically one such approach. Such editing involves site-specific digestion of mutant genomic DNA followed by repair. Previous experimental approaches were hampered by inaccurate and high rates of off-site lesioning and by overall low digestion rates. A new tool, clustered regularly interspaced short palindromic repeats coupled with the nuclease Cas9, may address both shortcomings. Some of the many challenges that must be addressed in moving clustered regularly interspaced short palindromic repeats coupled with the nuclease Cas9 therapies to the ophthalmic clinic are discussed here.
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Computational prediction of CRISPR cassettes in gut metagenome samples from Chinese type-2 diabetic patients and healthy controls.
BACKGROUND: CRISPR has been becoming a hot topic as a powerful technique for genome editing for human and other higher organisms. The original CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats coupled with CRISPR-associated proteins) is an important adaptive defence system for prokaryotes that provides resistance against invading elements such as viruses and plasmids. A CRISPR cassette contains short nucleotide sequences called spacers. These unique regions retain a history of the interactions between prokaryotes and their invaders in individual strains and ecosystems. One important ecosystem in the human body is the human gut, a rich habitat populated by a great diversity of microorganisms. Gut microbiomes are important for human physiology and health. Metagenome sequencing has been widely applied for studying the gut microbiomes. Most efforts in metagenome study has been focused on profiling taxa compositions and gene catalogues and identifying their associations with human health. Less attention has been paid to the analysis of the ecosystems of microbiomes themselves especially their CRISPR composition. RESULTS: We conducted a preliminary analysis of CRISPR sequences in a human gut metagenomic data set of Chinese individuals of type-2 diabetes patients and healthy controls. Applying an available CRISPR-identification algorithm, PILER-CR, we identified 3169 CRISPR cassettes in the data, from which we constructed a set of 1302 unique repeat sequences and 36,709 spacers. A more extensive analysis was made for the CRISPR repeats: these repeats were submitted to a more comprehensive clustering and classification using the web server tool CRISPRmap. All repeats were compared with known CRISPRs in the database CRISPRdb. A total of 784 repeats had matches in the database, and the remaining 518 repeats from our set are potentially novel ones. CONCLUSIONS: The computational analysis of CRISPR composition based contigs of metagenome sequencing data is feasible. It provides an efficient approach for finding potential novel CRISPR arrays and for analysing the ecosystem and history of human microbiomes.
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CRISPR Becomes Clearer.
In this pivotal year for gene editing, the breakthrough molecular system CRISPR-Cas9 has advanced on three fronts. In under seven months, an influential scientific body-the National Academies of Sciences, Engineering, and Medicine the National Academies of Sciences, Engineering, and Medicine-cracked open the door to human germline gene editing, ownership of patents covering CRISPR-Cas9 came into much sharper focus as a result of a dispute between two parties, and experiments showing proof of concept of the most controversial of uses-altering germlines of humans-were revealed as having been successfully performed by a mainstream laboratory. Given the vast spoils that await the patent owners, final results of all patent disputes over CRISPR-Cas9 patents may stretch on for years. Meanwhile, bioethical considerations of CRISPR-Cas9 have also been contentious as the United States and other countries grapple with how best to regulate gene editing.
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Precision medicine for sodium channelopathy-related autism and epilepsy.
Precision medicines for monogenic brain disorders are rapidly advancing. Voltage-gated sodium channel (VGSC) genes are the leading monogenic cause of severe epilepsy and profound autism spectrum disorder (ASD), most notably SCN1A, SCN2A, SCN3A, and SCN8A. Recent advances in animal and human induced pluripotent stem cell (hiPSC) disease models provide a powerful platform for advancing precision medicines. Thanks to the genomic revolution, many gene therapies are in preclinical studies and clinical trials for VGSC-related diseases, including viral vector gene replacement, clustered regularly interspaced short palindromic repeats (CRISPR) base editing, prime editing, and genetic modulation strategies including antisense oligonucleotides, engineered tRNAs, and CRISPR activation/interference (CRISPRa/i). This review highlights the latest advances in disease modeling and next-generation therapeutic development to advance precision medicine for VGSC-related brain disorders.
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Getting better all the time - recent progress in the development of CRISPR/Cas-based tools for plant genome engineering.
Since their first adaptation for plant genome editing, clustered regularly interspaced short palindromic repeats/CRISPR-associated system nucleases and tools have revolutionized the field. While early approaches focused on targeted mutagenesis that relies on mutagenic repair of induced double-strand breaks, newly developed tools now enable the precise induction of predefined modifications. Constant efforts to optimize these tools have led to the generation of more efficient base editors with enlarged editing windows and have enabled previously unachievable C-G transversions. Prime editors were also optimized for the application in plants and now allow to accurately induce substitutions, insertions, and deletions. Recently, great progress was made through precise restructuring of chromosomes, which enables not only the breakage or formation of genetic linkages but also the swapping of promoters.
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Fanconi anemia gene editing by the CRISPR/Cas9 system.
Genome engineering with designer nucleases is a rapidly progressing field, and the ability to correct human gene mutations in situ is highly desirable. We employed fibroblasts derived from a patient with Fanconi anemia as a model to test the ability of the clustered regularly interspaced short palindromic repeats/Cas9 nuclease system to mediate gene correction. We show that the Cas9 nuclease and nickase each resulted in gene correction, but the nickase, because of its ability to preferentially mediate homology-directed repair, resulted in a higher frequency of corrected clonal isolates. To assess the off-target effects, we used both a predictive software platform to identify intragenic sequences of homology as well as a genome-wide screen utilizing linear amplification-mediated PCR. We observed no off-target activity and show RNA-guided endonuclease candidate sites that do not possess low sequence complexity function in a highly specific manner. Collectively, we provide proof of principle for precision genome editing in Fanconi anemia, a DNA repair-deficient human disorder.
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