Cas9 Protein - Crossref

DNA-free genome editing in grapevine using CRISPR/Cas9 ribonucleoprotein complexes followed by protoplast regeneration

Although the direct delivery of Cas9-single guide RNA ribonucleoprotein complexes to grapevine protoplasts has been shown before, the regeneration of edited protoplasts into whole plants has yet to be revealed. Here, we outline an effective way to get transgene-free edited grapevine plants by the transfection and subsequent regeneration of protoplasts isolated from embryogenic callus. RNPs were directed from protoplasts to vines as proof of concept. Thompson Seedless, a single-copy green fluorescent protein reporter gene, was criticized and knocked out by direct delivery of RNPs. The transition of GFP-2112 protoplasts into whole plants was tracked throughout development, ensuring that the edited grapevine plants were similar in morphology and growth habit to wild-type controls. By the direct delivery of preassembled Cas9-sgRNA RNP complexes into protoplasts, we present the first highly effective method for DNA-free genome editing in grapevine, assisting in addressing regulatory issues pertaining to genetically modified plants.

Source link: https://doi.org/10.1093/hr/uhac240

CRISPR/Cas9-mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells

However, recent live cell superresolution microscopy studies tend to rely on the overexpression of a host protein fused to a fluorescent protein. Here, we demonstrate CRISPR/Cas9-mediated generation of heterozygous and homozygous human knockin cell lines expressing fluorescently tagged proteins from their respective native genomic loci at near-to-endogenous levels. We show sub-diffraction resolution images of living human knockin cells using low light intensity RESOLFT nanoscopy. Our effort to produce human cell lines expressing fluorescent fusion proteins at endogenous concentrations for RESOLFT nanoscopy can be extended to other fluorescent tags and super-resolution methods.

Source link: https://doi.org/10.1038/srep09592

Homology-directed repair in rodent zygotes using Cas9 and TALEN engineered proteins

Instead of mRNA, we hope to improve the quality of CRISPRs-Cas9 and TALENs homology-directed repair by inserting TALENs and Cas9 proteins, rather than mRNA, microinjected into rat and mouse zygotes, along with long or short donor DNAs. We found that Cas9 protein was more effective at homology-directed repair than mRNA, while TALEN protein was less effective than mRNA at initiating homology-directed repair, although mRNA was less effective than mRNA. Our results show that Cas9 protein may be a convenient and cost-effective therapeutic alternative to Cas9 mRNA in the generation of genetically modified rats and mice, as well as other mammals.

Source link: https://doi.org/10.1038/srep14410

Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos

We observed fewer off-target effects of Cas9 and more point mutation knock-in efficiency through injection of Cas9 protein rather than mRNA into embryos, as well as increased point mutation knock-in efficiency. Large genomic DNA fragment deletion mice were developed for in vitro study of lncRNAs and gene clusters. The introduction of a 2. 7 kb CreERT2 cassette into the mouse Nfatc1 locus enabled identification and tracing of hair follicle stem cells.

Source link: https://doi.org/10.1038/srep17517

Fluorescent protein tagging of endogenous protein in brain neurons using CRISPR/Cas9-mediated knock-in and in utero electroporation techniques

Abstract Genome editing is a simple method for investigating gene functions. Here, we were able to code EGFP-tagged peptide in neural neurons in the brain immediately after the first ATG codon of the b2-actin gene in neurons in the brain by the combined use of the CRISPR/Cas9 system and in utero electroporation technique, resulting in the expression of the EGFP-tagged u03b2-actin protein in cortical layer 2/3 pyramidal neurons. In the soma and neurites of EGFP knock-in neurons, we found EGFP fluorescence signals. No apparent changes in spine density nor basic electrophysiological properties were found in EGFP knock-in neurons.

Source link: https://doi.org/10.1038/srep35861

DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins

Abstract Microalgae are multicellular organisms capable of converting CO 2, H 2 O, and sunlight into fuel and chemicals for domestic and industrial use. Here we present a one-step change of Chlamydomonas reinhardtii by the DNA-free CRISPR-Cas9 procedure rather than plasmids that encode Cas9 and guide RNAs.

Source link: https://doi.org/10.1038/srep30620

RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses

We're discussing one-step cloning of gRNA oligonucleotides and the rapid development of short-arm targeting constructs with the ability of Cas9-assisted efforts to establish protein tagged alleles in embryonic stem cells at high rate. With the auxin degron, we are focusing on protein tagging because it is a ligand-controlled loss-of-function scheme that is both accurate and reversible. Despite high incidences of monoallelic RAC-targeting, we found that simultaneous biallelic targeting gains from long-arm targeting techniques were evidently beneficial.

Source link: https://doi.org/10.1038/srep25529

Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes

Abstract: Targeted DNA double-strand breaks have been shown to greatly increase the frequency and precision of genome editing's transcription. Currently, genome editing in plants primarily depends on the delivery of double-strand break reagents in the form of DNA vectors. Both mutated and edited alleles are present in this report. We report on the biolistic assembly of pre-assembled Cas9u2013gRNA ribonucleoproteins into maize embryo cells and plant regeneration.

Source link: https://doi.org/10.1038/ncomms13274

A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A

Here, we created a protein stability regulators screening assay by combining the whole-genome CRISPR-Cas9 library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to test for protein stability regulators. Cul4B-DDB1 DCAF8, which was used as an example by Cdc25A, was identified as a new E3 ligase for Cdc25A. This is the first study to show that acetylation, as a novel post-translational modification, boosts Cdc25A stability, and we suggest that this unbiased CRISPR-Cas9 screening procedure at the genome scale can be used to globally identify protein stability regulators.

Source link: https://doi.org/10.1038/celldisc.2016.14

CRISPR Cas9 ribonucleoprotein-mediated genomic editing in primary innate immune cells

Although decades of research have sought to genetically reprogram innate immunity for therapeutic uses, current therapies are often ineffective or nonspecific, restricting their use in primary innate immune cells. In addition, we describe in vivo adoptive transfer mouse models that can be used to screen for gene function during viral infection by using cRNP-edited nau00efve NK cells and bone marrow-derived dendritic cell precursors. This universally applicable strategy would improve target gene discovery and provide a specific and simplified route to gene editing in the innate immune system.

Source link: https://doi.org/10.4049/jimmunol.204.supp.159.3

* Please keep in mind that all text is summarized by machine, we do not bear any responsibility, and you should always check original source before taking any actions