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Assembly of Baculovirus Vectors for Multiplexed Prime Editing.
Efficient genome editing by using CRISPR technologies requires simultaneous and efficient delivery of multiple genetically encoded components to mammalian cells. Amongst all editing approaches, prime editing (PE) has the unique potential to perform seamless genome rewriting, in the absence of DNA double-strand breaks (DSBs). The cargo capacity required for efficient PE delivery to mammalian cells stands at odd with the limited packaging capacity of traditional viral delivery vectors. By contrast, baculovirus (BV) has a large synthetic DNA capacity and can efficiently transduce mammalian cells. Here we describe a protocol for the assembly of baculovirus vectors for multiplexed prime editing in mammalian cells.
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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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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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From bacterial battles to CRISPR crops; progress towards agricultural applications of genome editing.
Genome editing is the precise alteration of DNA in living cells by the cutting or removal of specific sequences, sometimes followed by insertion of new sequences at the cut site. CRISPR-Cas9 has become firmly established as the genome-editing method of choice, replacing the systems that had been developed and in use since the early 1990s. The CRISPR-Cas9 system has been developed from a mechanism used in prokaryotes as a defence against bacteriophage but actually functions in cells of all types of organisms. It is widely used in research as a gene knockout and editing tool; applications in veterinary medicine (such as increased resistance to disease) and human medicine (such as correction of disease-causing mutations) are under development. In agriculture and horticulture, the potential for various aspects of crop improvement is very large. Selected aspects of this potential are presented here, with particular focus on crop quality and disease resistance. The article ends with a brief discussion of the regulatory 'environment' in the USA and the EU.
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Conditional Expression of Cas9 and dCas9 in Lucilia cuprina Reveals dCas9-Associated Lethality.
Conditional sex transformation systems could improve genetic control strategies against insect pests. Here, we developed and tested CRISPR-based, tetracycline-repressible sex transformation strains in the Australian sheep blowfly, Lucilia cuprina. Using Tet-Off-regulated expression of Cas9 and dCas9, we targeted the sex-determining gene transformer with the goal of converting females into males. Conditional Cas9 expression enabled knockout of a visual marker gene, confirming inducible genome editing. However, strains expressing transformer-targeting sgRNA arrays did not undergo sex transformation. Embryonic microinjection of transformer-targeting sgRNAs into Cas9-expressing embryos produced intersex individuals, indicating that sgRNA expression from the integrated arrays was insufficient to disrupt the sex determination pathway. In contrast, high-level dCas9 expression was associated with developmental delays, reduced body weight, and lethality. These findings establish the first conditional CRISPR expression system in L. cuprina and demonstrate that Cas9 is compatible with inducible gene editing, whereas dCas9 is poorly tolerated at high expression levels.
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Recent advances in globin research using genome-wide association studies and gene editing.
A long-sought goal in the hemoglobin field has been an improved understanding of the mechanisms that regulate the switch from fetal (HbF) to adult (HbA) hemoglobin during development. With such knowledge, the hope is that strategies for directed reactivation of HbF in adults could be devised as an approach to therapy for the β-hemoglobinopathies thalassemia and sickle cell disease. Recent genome-wide association studies (GWAS) led to identification of three loci (BCL11A, HBS1L-MYB, and the β-globin cluster itself) in which natural genetic variation is correlated with different HbF levels in populations. Here, the central role of BCL11A in control of HbF is reviewed from the perspective of how findings may be translated to gene therapy in the not-too-distant future. This summary traces the evolution of recent studies from the initial recognition of BCL11A through GWAS to identification of critical sequences in an enhancer required for its erythroid-specific expression, thereby highlighting an Achilles heel for genome editing.
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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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A homology independent sequence replacement strategy in human cells using a CRISPR nuclease.
Precision genomic alterations largely rely on homology directed repair (HDR), but targeting without homology using the non-homologous end-joining (NHEJ) pathway has gained attention as a promising alternative. Previous studies demonstrated precise insertions formed by the ligation of donor DNA into a targeted genomic double-strand break in both dividing and non-dividing cells. Here, we demonstrate the use of NHEJ repair to replace genomic segments with donor sequences; we name this method 'Replace' editing (Rational end-joining protocol delivering a targeted sequence exchange). Using CRISPR/Cas9, we create two genomic breaks and ligate a donor sequence in-between. This exchange of a genomic for a donor sequence uses neither microhomology nor homology arms. We target four loci in cell lines and show successful exchange of exons in 16-54% of human cells. Using linear amplification methods and deep sequencing, we quantify the diversity of outcomes following Replace editing and profile the ligated interfaces. The ability to replace exons or other genomic sequences in cells not efficiently modified by HDR holds promise for both basic research and medicine.
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Single-nucleotide-level mapping of DNA regulatory elements that control fetal hemoglobin expression.
Pinpointing functional noncoding DNA sequences and defining their contributions to health-related traits is a major challenge for modern genetics. We developed a high-throughput framework to map noncoding DNA functions with single-nucleotide resolution in four loci that control erythroid fetal hemoglobin (HbF) expression, a genetically determined trait that modifies sickle cell disease (SCD) phenotypes. Specifically, we used the adenine base editor ABEmax to introduce 10,156 separate A•T to G•C conversions in 307 predicted regulatory elements and quantified the effects on erythroid HbF expression. We identified numerous regulatory elements, defined their epigenomic structures and linked them to low-frequency variants associated with HbF expression in an SCD cohort. Targeting a newly discovered γ-globin gene repressor element in SCD donor CD34+ hematopoietic progenitors raised HbF levels in the erythroid progeny, inhibiting hypoxia-induced sickling. Our findings reveal previously unappreciated genetic complexities of HbF regulation and provide potentially therapeutic insights into SCD.
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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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