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Electroporation-Mediated CRISPR/Cas9 Genome Editing in Rat Zygotes.
Genetic engineering in the rat has been revolutionized by the development of CRISPR-based genome editing tools. Conventional methods for inserting genome editing elements such as CRISPR/Cas9 reagents into rat zygotes include cytoplasmic or pronuclear microinjections. These techniques are labor-intensive, require specialized micromanipulator equipment, and are technically challenging. Here, we describe a simple and effective method for zygote electroporation in which CRISPR/Cas9 reagents are introduced into rat zygotes via pores produced by precise electrical pulses applied to the cells. Zygote electroporation allows for high-throughput efficient genome editing in rat embryos.
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Giving Genome Editing the Fingers: An Interview with Dana Carroll.
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Targeted Gene Editing in Wheat During Haploid Production via Wide Hybridization with Transgenic Maize Expressing Cas9 and Guide RNA.
The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system is an efficient and versatile genome engineering tool, which has been widely used for targeted mutagenesis and gene functional characterization in various organisms. This system is simple because it only requires a Cas9 enzyme serving as a nuclease and guide RNA (gRNA) containing a 20-nt sequence matching the target gene. Delivery of a vector expressing Cas9 and gRNA or the preassembled Cas9/gRNA complex as a ribonucleoprotein (RNP) into plant cells for gene targeting are usually via the biolistic- or Agrobacterium-mediated approach. However, most wheat genotypes suffer from low efficiency of callus induction and plant regeneration from explants receiving the vector or RNP delivered by the biolistic- or Agrobacterium-mediated transformation method, limiting the application of genome editing systems in many commercially grown wheat varieties. Here, we describe a stepwise protocol for targeted gene editing in wheat via wide hybridization with transgenic maize expressing Cas9 and gRNA. A binary vector expressing Cas9 and gRNA is constructed and used for Agrobacterium-mediated transformation to generate transgenic maize plants, which are used to pollinate emasculated spikes of wheat varieties. After fertilization, the maize chromosomes enter the transient hybrid zygote and the transgene (T-DNA) on a maize chromosome expresses the Cas9 enzyme and gRNA, which forms an RNP complex to edit the target gene in wheat genome. After several cell divisions, maize chromosomes in the hybrid zygote are eliminated, resulting in formation of haploid wheat embryos with the target gene edited, which can be rescued by embryo culture technique to produce haploid plants. Doubled haploid (DH) wheat plants with homozygous gene mutations are developed by chromosome doubling through colchicine treatment of the haploid plants. The wheat × maize hybridization combined with the CRISPR/Cas9 system provides a one-step approach for generating DH lines with the target gene edited from any wheat genotypes of interest.
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Precision gene editing technology and applications in nephrology.
The expanding field of precision gene editing is empowering researchers to directly modify DNA. Gene editing is made possible using synonymous technologies: a DNA-binding platform to molecularly locate user-selected genomic sequences and an associated biochemical activity that serves as a functional editor. The advent of accessible DNA-targeting molecular systems, such as zinc-finger nucleases, transcription activator-like effectors (TALEs) and CRISPR-Cas9 gene editing systems, has unlocked the ability to target nearly any DNA sequence with nucleotide-level precision. Progress has also been made in harnessing endogenous DNA repair machineries, such as non-homologous end joining, homology-directed repair and microhomology-mediated end joining, to functionally manipulate genetic sequences. As understanding of how DNA damage results in deletions, insertions and modifications increases, the genome becomes more predictably mutable. DNA-binding platforms such as TALEs and CRISPR can also be used to make locus-specific epigenetic changes and to transcriptionally enhance or suppress genes. Although many challenges remain, the application of precision gene editing technology in the field of nephrology has enabled the generation of new animal models of disease as well as advances in the development of novel therapeutic approaches such as gene therapy and xenotransplantation.
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Navigating gene editing in porcine embryos: Methods, challenges, and future perspectives.
Gene editing technologies, particularly CRISPR/Cas9, have emerged as transformative tools in genetic modification, significantly advancing the use of porcine embryos in biomedical and agricultural research. This review comprehensively examines the various methodologies for gene editing and delivery methods, such as somatic cell nuclear transfer (SCNT), microinjection, electroporation, and lipofection. This review, focuses on the advantages or limitations of using different biological sources (in vivo- vs. in vitro oocytes/embryos). Male germ cell manipulation using sperm-mediated gene transfer (SMGT) and testis-mediated gene transfer (TMGT) represent innovative approaches for producing genetically modified animals. Although these technologies have revolutionized the genetic engineering field, all these strategies face challenges, including high rates of off-target events and mosaicism. This review emphasizes the need to refine these methods, with a focus on reducing mosaicism and improving editing accuracy. Further advancements are essential to unlocking the full potential of gene editing for both agricultural applications and biomedical innovations.
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The protocol of tagging endogenous proteins with fluorescent tags using CRISPR-Cas9 genome editing.
The currently widely used CRISPR-Cas9 genome editing technology enables the editing of target genes (knock-out or knock-in) with high accuracy and efficiency. Guided by the small guide RNA, the Cas9 nuclease induces a DNA double-strand break at the targeted genomic locus. The DNA double-strand break can be repaired by the homology-directed repair pathway in the presence of a repair template. With the repair template containing the coding sequence of a fluorescent tag, the targeted gene can be inserted with the sequence of a fluorescent tag at the designed position. The genome editing mediated labeling of endogenous proteins with fluorescent tags avoids the potential artifacts caused by gene overexpression and substantially improves the reproductivity of imaging experiments. This protocol focuses on creating mammalian cell lines with endogenous proteins tagged with fluorescent proteins or self-labeling protein tags using CRISPR-Cas9 genome editing.
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Inducible CRISPR genome-editing tool: classifications and future trends.
The discovery of CRISPR-Cas9/dCas9 system has reinforced our ability and revolutionized our history in genome engineering. While Cas9 and dCas9 are programed to modulate gene expression by introducing DNA breaks, blocking transcription factor recruitment or dragging functional groups towards the targeted sites, sgRNAs determine the genomic loci where the modulation occurs. The off-target problem, due to limited sgRNA specificity and genome complexity of many species, has posed concerns for the wide application of this revolutionary technique. To solve this problem and, more importantly, gain power over gene functionality and cell fate control, inducible strategies have been continuously evolved to offer tailored solutions to address specific biological questions. By reviewing recent advances in inducible CRISPR system design and critical elements potentially adding values to such systems, we classify current approaches in this domain into four mechanically distinct categories, namely, "split system", "allosteric system", "combinatorial system", and "transient delivery system", discuss the pros and cons of each system, and point out the under-explored areas and future directions, with the aim of enriching our toolbox of delicate life engineering.
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CRISPR/Cas9: An RNA-guided highly precise synthetic tool for plant genome editing.
CRISPR/Cas9 is a newly developed and naturally occurred genome editing tool, which is originally used by bacteria for immune defence. In the past years, it has been quickly employed and modified to precisely edit genome sequences in both plants and animals. Compared with the well-developed zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 has lots of advantages, including easier to design and implement, higher targeting efficiency, and less expensive. Thus, it is becoming one of the most powerful tools for knockout of an individual gene as well as insertion of one gene and/or control of gene transcription. Studies have shown that CRISPR/Cas9 is a great tool to edit many genes in a variety of plant species, including the model plant species as well as agriculturally important crops, such as cotton, maize, wheat, and rice. CRISPR/Cas9-based genome editing can be used for plant functional studies and plant improvement to yield, quality, and tolerance to environmental stress.
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High-Throughput Screening of a Lipid Nanoparticle (LNP) Library Identifies Lead Formulations for CRISPR-Cas9 Ribonucleoprotein Delivery and Gene Editing.
Delivery of CRISPR-Cas9 ribonucleoprotein (RNP) using synthetic materials can bypass the host translational machinery and limit the duration of genomic exposure, minimizing the risk of off-target mutations. Despite their advantages, lipid nanoparticle (LNP) platforms for CRISPR-Cas9 RNP delivery that achieve highly efficient editing at low doses remain limited. In the present study, we developed a library of ionizable lipids (ILs) and corresponding LNPs to enhance the delivery and editing efficiencies of CRISPR-Cas9 RNP complexes. Following comprehensive in vitro screening and formulation optimization, an optimal LNP candidate containing the C12A9 IL (denoted as LNPC12A9) was identified that achieved ∼90% EGFP disruption in HEK293T-EGFP cells at an RNP concentration of 30 nM and ∼60% at 15 nM. The editing performance was consistently observed at an RNP concentration of 15 nM when editing the Gcgr and HPRT1 genes across multiple hepatic cell lines. Moreover, coencapsulation of RNP and a single-stranded oligodeoxynucleotide (ssODN) template for homology-directed repair (HDR) within LNPC12A9 induced an HDR rate of 37.8%. Systemic administration of LNPC12A9 into C57BL/6J mice led to liver tropism and a total hepatic Pcsk9 gene disruption rate of 1.39%. In conclusion, this study provides insights into the construction of an LNP platform to improve the delivery efficiency of CRISPR-Cas9 RNP, thereby facilitating the development of LNPs for safe, precise, and tissue-specific Cas9 RNP-based genome editing therapies.
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Gene editing and genetic engineering approaches for advanced probiotics: A review.
The applications of probiotics are significant and thus resulted in need of genome analysis of probiotic strains. Various omics methods and systems biology approaches enables us to understand and optimize the metabolic processes. These techniques have increased the researcher's attention towards gut microbiome and provided a new source for the revelation of uncharacterized biosynthetic pathways which enables novel metabolic engineering approaches. In recent years, the broad and quantitative analysis of modified strains relies on systems biology tools such as in silico design which are commonly used methods for improving strain performance. The genetic manipulation of probiotic microorganisms is crucial for defining their role in intestinal microbiota and exploring their beneficial properties. This review describes an overview of gene editing and systems biology approaches, highlighting the advent of omics methods which allows the study of new routes for studying probiotic bacteria. We have also summarized gene editing tools like TALEN, ZFNs and CRISPR-Cas that edits or cleave the specific target DNA. Furthermore, in this review an overview of proposed design of advanced customized probiotic is also hypothesized to improvise the probiotics.
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