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No apparent p53 activation in CRISPR-engineered gene-edited rabbits.
Clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9) and base editors (BEs) are revolutionary gene-editing technology that has been widely utilized in biology, biotechnology and medicine. However, recent reports show that CRISPR-Cas9-mediated genome editing can induce a p53-mediated stress response and cell cycle arrest in human cells, while not illustrated in gene-editing animals. In the study, to verify whether there is a phenomenon of p53 activation, by analysing nine gene-edited rabbits using CRISPR-Cas9 and BEs, we provide the first evidence that no apparent p53 expression changes in those rabbits generated by Cas9 or BE-edited, suggesting that p53 may not need to consider for application in gene-edited animals.
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Cell-Penetrating Peptides and CRISPR-Cas9: A Combined Strategy for Human Genetic Disease Therapy.
The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated nuclease 9 (Cas9) technology has revolutionized the field of genetic engineering, offering unprecedented potential for the targeted manipulation of DNA sequences. Advances in the mechanism of action of the CRISPR-Cas9 system allowed potential applicability for the treatment of genetic diseases. CRISPR-Cas9's mechanism of action involves the use of an RNA guide molecule to target-specific DNA sequences and the Cas9 enzyme to induce precise DNA cleavage. In the context of the CRISPR-Cas9 system, this review covers nonviral delivery methods for gene editing based on peptide internalization. Here, we describe critical areas of discussion such as immunogenicity, emphasizing the importance of safety, efficiency, and cost-effectiveness, particularly in the context of treating single-mutation genetic diseases using advanced editing techniques genetics as prime editor and base editor. The text discusses the versatility of cell-penetrating peptides (CPPs) in forming complexes for delivering biomolecules, particularly ribonucleoprotein for genome editing with CRISPR-Cas9 in human cells. In addition, it emphasizes the promise of combining CPPs with DNA base editing and prime editing systems. These systems, known for their simplicity and precision, hold great potential for correcting point mutations in human genetic diseases. In summary, the text provides a clear overview of the advantages of using CPPs for genome editing with CRISPR-Cas9, particularly in conjunction with advanced editing systems, highlighting their potential impact on clinical applications in the treatment of single-mutation genetic diseases. [Figure: see text].
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Genome editing: An insight into disease resistance, production efficiency, and biomedical applications in livestock.
One of the primary concerns for the survival of the human species is the growing demand for food brought on by an increasing global population. New developments in genome-editing technology present promising opportunities for the growth of wholesome and prolific farm animals. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. Genome editing entails modifying genetic material by removing, adding, or manipulating particular DNA sequences from a particular locus in a way that does not happen naturally. The three primary genome editors are CRISPR/Cas 9, TALENs, and ZFNs. Each of these enzymes is capable of precisely severing nuclear DNA at a predetermined location. One of the most effective inventions is base editing, which enables single base conversions without the requirement for a DNA double-strand break (DSB). As reliable methods for precise genome editing in studies involving animals, cytosine and adenine base editing are now well-established. Effective zygote editing with both cytosine and adenine base editors (ABE) has resulted in the production of animal models. Both base editors produced comparable outcomes for the precise editing of point mutations in somatic cells, advancing the field of gene therapy. This review focused on the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of ZFNs, TALENs, and CRISPR/Cas9 base editors, and prime editing in diverse lab and farm animals. Additionally, we address the methodologies that can be used for gene regulation, base editing, and epigenetic alterations, as well as the significance of genome editing in animal models to better reflect real disease. We also look at methods designed to increase the effectiveness and precision of gene editing tools. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. This review is an overview of the existing knowledge of the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of zinc finger nucleases (ZFNs), transcription-activator-like endonucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas 9), base editors and prime editing in diverse lab and farm animals, which will offer better and healthier products for the entire human race.
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Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Gene Editing Technique in Xenotransplantation.
Genetically modified pigs have been considered favorable resources in xenotransplantation. Microinjection of randomly integrating transgenes into zygotes, somatic cell nuclear transfer, homologous recombination, zinc finger nucleases, transcription activator-like effector nucleases, and most recently, clustered regularly interspaced short palindromic repeats-cas9 (CRISPR/Cas9) are the techniques that have been used to generate these animals. Here, we provide an overview of the CRISPR approaches that have been used to modify genes which are vital in improving xenograft survival rate, including cytidine monophosphate-N-acetylneuraminic acid hydroxylase, B1,4N-acetylgalactosaminyltransferase, isoglobotrihexosylceramide synthase, class I MHC, von Willebrand factor, C3, and porcine endogenous retroviruses. In addition, we will mention the importance of potential candidate genes which could be targeted using CRISPR/Cas9.
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CRISPR: gene editing is just the beginning.
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CRISPR/Cas Derivatives as Novel Gene Modulating Tools: Possibilities and In Vivo Applications.
The field of genome editing started with the discovery of meganucleases (e.g., the LAGLIDADG family of homing endonucleases) in yeast. After the discovery of transcription activator-like effector nucleases and zinc finger nucleases, the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) system has opened a new window of applications in the field of gene editing. Here, we review different Cas proteins and their corresponding features including advantages and disadvantages, and we provide an overview of the different endonuclease-deficient Cas protein (dCas) derivatives. These dCas derivatives consist of an endonuclease-deficient Cas9 which can be fused to different effector domains to perform distinct in vitro applications such as tracking, transcriptional activation and repression, as well as base editing. Finally, we review the in vivo applications of these dCas derivatives and discuss their potential to perform gene activation and repression in vivo, as well as their potential future use in human therapy.
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Core Concept: CRISPR gene editing.
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CRISPR/Cas: A Faster and More Efficient Gene Editing System.
Gene editing technology has been at its mature stage with the successful development of TALENs and CRISPR/Cas enzymes. The genetically modified endonucleases of ZFNs, TALENs, and CRISPR/Cas are widely used in the development of genetically modified cells or organisms. Among the enzymes that possess gene editing ability, CRISPR/Cas is the latest member with high efficiency in gene editing and simplicity in cloning. This review discusses the discovery of CRISPR, the development of the CRISPR/Cas system, and its applications as a new gene editing system.
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CRISPR-Cas14 is now part of the artillery for gene editing and molecular diagnostic.
Recently Jennifer Doudna's group discovered the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 14 (Cas14), identified almost exclusively in a superphylum of extremophile archaea. The newly discovered Cas14 possesses a single-stranded (ss)DNA targeting activity - despite being two times smaller than Cas9 - a capability that might confer a defense against viruses with ssDNA genomes. Furthermore, by combining the non-specific ssDNase cleavage activity of Cas14 with isothermal amplification method (DETECTR-Cas14), it can also be promisingly exploited for high-fidelity DNA single-nucleotide polymorphism genotyping, and potentially for detecting ssDNA viruses of undeniable clinical, ecological, and economic importance infecting hosts in all three domains of life. Thus, CRISPR-Cas14 might acquire an exponential expansion in the field of CRISPR diagnostic for infectious and noninfectious diseases.
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High-efficiency genome editing by Cas12a ribonucleoprotein complex in Euglena gracilis.
Transgene-free genome editing based on clustered regularly interspaced short palindromic repeats (CRISPR) technology is key to achieving genetic engineering in microalgae for basic research and industrial applications. Euglena gracilis, a unicellular phytoflagellate microalga, is a promising biomaterial for foods, feeds, cosmetics and biofuels. However, methods for the genetic manipulation of E. gracilis are still limited. Here, we developed a high-efficiency, transgene-free genome editing method for E. gracilis using Lachnospiraceae bacterium CRISPR-associated protein 12a (LbCas12a) ribonucleoprotein (RNP) complex, which complements the previously established Cas9 RNP-based method. Through the direct delivery of LbCas12a-containing RNPs, our method reached mutagenesis rates of approximately 77.2-94.5% at two different E. gracilis target genes, Glucan synthase-like 2 (EgGSL2) and a phytoene synthase gene (EgcrtB). Moreover, in addition to targeted mutagenesis, we demonstrated efficient knock-in and base editing at the target site using LbCas12a-based RNPs with a single-stranded DNA donor template in E. gracilis. This study extends the genetic engineering capabilities of Euglena to accelerate its basic use for research and engineering for bioproduction.
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