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Found: 26 168
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A novel in vivo genome editing doubled haploid system for Zea mays L.
Doubled haploid (DH) technologies accelerate maize inbred development. Recently, methods using CRISPR-Cas have created gene-edited maize DH populations, albeit with relatively low editing frequencies. Restoring fertility via haploid chromosome doubling remains a critically important production constraint. Thus, improved editing and chromosome doubling outcomes are needed. Here we obtained maternally derived diploid embryos in vivo by ectopically co-expressing Zea mays BABY BOOM and cyclin D-like gene products within unfertilized egg cells. When combined with gene editing, the in vivo method enables the production of mature seed with a maternally derived, gene-edited diploid embryo without requiring in vitro tissue culture methods nor the use of a chemical chromosome doubling agent. In summary, we report a novel approach for creating gene-edited maize DH populations that we expect can accelerate genetic gain in a scalable, cost-effective manner.
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The application of CRISPR/Cas9 in genome editing of filamentous fungi.
Filamentous fungi usually refer to the eukaryotic microorganisms with developed mycelia and without large fruiting bodies. Filamentous fungi not only play an important role in material cycle in nature, but also are closely related to human health, industry and agriculture. However, the difficulties in genetic manipulation impede the molecular studies on filamentous fungi. Clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) is a conservative immune defense mechanism found in bacteria and archaea. Recently, it has been developed as a convenient and flexible technique for genome editing. At present, CRISPR/Cas9 has been applied in different species for genetic operation. This review summarizes the application of CRISPR/Cas9 in genome editing of filamentous fungi and aims to provide a reference for the research in this field.
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CRISPR-Activation: Boosting Expression of Plant MIRs.
In the field of plant genome editing, the CRISPR/Cas system stands out as an exceptionally versatile tool. Originally conceived for gene editing, the catalytically inactive Cas9 variant, known as dead Cas9 (dCas9), takes on a new role when coupled with transcriptional effectors, offering a finely tuned mechanism for controlling gene expression. This innovation has paved the way for the evolution of sophisticated systems such as CRISPR-Act 2.0 and CRISPR-Act 3.0, which significantly enhance transcriptional activation in plants. This chapter serves as an introduction to a protocol that harnesses CRISPR-activation (CRISPRa) to modulate the transcription of plant miRNA genes. Recognizing the imperative for meticulous construct design and promoter identification, the protocol outlines steps encompassing vector construction. It underscores the critical importance of comprehending miRNA promoter regions and cis-acting elements, presenting a holistic approach for effectively employing CRISPRa in manipulating miRNA expression to modulate traits in plants.
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CRISPR-Cpf1-Mediated T4 Phage Genome Editing for One-Step In Vivo Display of Heterologous Protein.
The T4 phage is a robust vector for high-density heterologous protein display. It leverages two non-essential outer capsid proteins, i.e., Soc (~870 copies) and Hoc (~155 copies). These two proteins enable the efficient display of target proteins on the capsid of T4. Here, we detail the workflow for one-step in vivo display of a heterologous protein. Specifically, this method utilizes CRISPR-Cpf1-mediated gene editing technology to insert the sequence of interest (using mCherry as an example) downstream of the Soc encoding gene within the T4 phage genome, resulting in a Soc-fused recombinant protein. This engineering method allows for endogenous expression of the Soc-mCherry recombinant protein within Escherichia coli cells during phage replication, after which the recombinant protein spontaneously assembles onto the capsid of the engineered phage. By providing a universal framework suitable for in vivo display, this approach empowers researchers to readily construct tailored T4 nanoparticles for diverse biotechnological applications.
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Seeking more nucleases.
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CRISPR/Cas9 applications in gene therapy for primary immunodeficiency diseases.
Primary immunodeficiency diseases (PIDs) encompass a range of diseases due to mutations in genes that are critical for immunity. Haploinsufficiency and gain-of-function mutations are more complex than simple loss-of-function mutations; in addition to increased susceptibility to infections, immune dysregulations like autoimmunity and hyperinflammation are common presentations. Hematopoietic stem cell (HSC) gene therapy, using integrating vectors, provides potential cure of disease, but genome-wide transgene insertions and the lack of physiological endogenous gene regulation may yet present problems, and not applicable in PIDs where immune regulation is paramount. Targeted genome editing addresses these concerns; we discuss some approaches of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system applicable for gene therapy in PIDs. Preclinical repair of gene mutations and insertion of complementary DNA restore endogenous gene regulation and they have shown very promising data for clinical application. However, ongoing studies to characterize off-target genotoxicity, careful donor designs to ensure physiological expression, and maneuvers to optimize engraftment potential are critical to ensure successful application of this next-gen targeted HSC gene therapy.
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Plasmodium falciparum In Vitro Drug Resistance Selections and Gene Editing.
Malaria continues to be a global health burden, threatening over 40% of the world's population. Drug resistance in Plasmodium falciparum, the etiological agent of the majority of human malaria cases, is compromising elimination efforts. New approaches to treating drug-resistant malaria benefit from defining resistance liabilities of known antimalarial agents and compounds in development and defining genetic changes that mediate loss of parasite susceptibility. Here, we present protocols for in vitro selection of drug-resistant parasites and for site-directed gene editing of candidate resistance mediators to test for causality.
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A New Generation of Lineage Tracing Dynamically Records Cell Fate Choices.
Reconstructing the development of lineage relationships and cell fate mapping has been a fundamental problem in biology. Using advanced molecular biology and single-cell RNA sequencing, we have profiled transcriptomes at the single-cell level and mapped cell fates during development. Recently, CRISPR/Cas9 barcode editing for large-scale lineage tracing has been used to reconstruct the pseudotime trajectory of cells and improve lineage tracing accuracy. This review presents the progress of the latest CbLT (CRISPR-based Lineage Tracing) and discusses the current limitations and potential technical pitfalls in their application and other emerging concepts.
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Age-related cardiopathies gene editing.
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Guanidinium-Rich Lipopeptide-Based Nanoparticle Enables Efficient Gene Editing in Skeletal Muscles.
Genome editing mediated by the CRISPR-Cas system holds great promise for the treatment of genetic diseases. However, safe and efficient in vivo delivery of CRISPR genome editing machinery remains a challenge. Here, we report a lipopeptide-based nanoparticle (LNP) that can efficiently deliver the CRISPR Cas9/sgRNA ribonucleoprotein (RNP) and enable efficient genome editing both in vitro and in vivo. An artificial lipopeptide, GD-LP, was constructed by linking a hydrophilic guanidinium-rich head to an oleic acid-based hydrophobic tail via a disulfide bond. LNP formed by the self-assembly of GD-LP can easily form a complex with RNP with a loading content of up to 20 wt %. The resulting RNP-LNP nanocomplex led to 72.6% gene editing efficiency in GFP-HEK cells with negligible cytotoxicity. The LNP also showed significantly higher transfection efficiencies than Lipofectamine 2000 for the delivery of mRNA in NIH 3T3 and RAW 264.7 and the delivery of plasmid DNA in B78 cells. In vivo studies showed that intramuscular injection of the RNP-LNP nanocomplex in Ai14 mice induced efficient gene editing in muscular tissues. Moreover, the delivery of Cas9 RNP and donor DNA by LNP (i.e., RNP/ssODN-LNP nanocomplex) restored dystrophin expression, reduced skeletal muscle fibrosis, and significantly improved muscle strength in a Duchenne muscular dystrophy (DMD) mouse model.
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