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Cas9 - Europe PMC

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Last Updated: 10 January 2023

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DUSP2 deletion with CRISPR/Cas9 promotes Mauthner cell axonal regeneration at the early stage of zebrafish.

In zebrafish with spinal cord injury, dusp2 was identified as upregulated gene. A new analysis showed that it was not a candidate for spinal cord injury. We found that dual specificity phosphatase 2 in this case is a negative regulator of axon regeneration of the Mauthner cell. In this research, we found that M-cell axons of dusp2 had a faster recovery at the early stage after birth, when those of dusp2+/- zebrafish did not. Overexpression of DUSP2 in Tg zebrafish by single-cell electroporation has slowed M-cell electroporation and delayed the cell regeneration.

Source link: https://europepmc.org/article/MED/36018180


CRISPR-Cas9 recognition of enzymatically synthesized base-modified nucleic acids.

Furthermore, dthG could be integrated into PCR samples by Taq DNA polymerase together with the other three base-modified nucleotides. In an in vitro CRISPR-Cas9 cleavage assay, the obtained RNA products containing thG, as well as thG, along with 5-bromocytosine may perform as well as 5-bromocytosine. When included in sgRNA, the N1-Methylpseudouridine was also shown to be a faithful non-canonical substitute for uridine to direct Cas9 nuclease cleavage. The Cas9 inactivation by 7-deazapurines showed the importance of the 7-nitrogen atom of purines in both sgRNA and PAM sites for achieving effective Cas9 cleavage.

Source link: https://europepmc.org/article/MED/36611237


Clustered regularly interspaced short palindromic repeats/Cas9-mediated gene editing. A promising strategy in hematological disorders.

In fact, very promising treatment of hematological disorders have been developed in recent years, based on the self-renewal and multipotent differentiation properties of hematopoietic stem and progenitor cells, which make this cell subset the ideal target for gene therapy purposes. We can therefore utilize CRISPR/Cas9's various DNA repair techniques, from homology-directed repair to non-homologous-joining to the most recent emerging technologies such as base and prime editing, considering the variety of genes and mutations involved. Although the delivery systems for hematopoietic stem and progenitor cells are still the bottleneck of this technology, several of the genetic editing advances in this review have already hit a clinical stage and yielded promising preliminary results.

Source link: https://europepmc.org/article/MED/36610813


Generation of eco-friendly channel catfish, Ictalurus punctatus , harboring alligator cathelicidin gene with robust disease resistance by harnessing different CRISPR/Cas9-mediated systems

To minimize introgression of transgenic animals as transgenic animals, catfish species have been tested for improved disease resistance and interference in reproduction to minimize introgression of escapees and control reproduction. We succeeded in integrating the cathelicidin gene from an alligator into the target luteinizing hormone locus of channel catfish using two delivery methods assisted by double-stranded DNA and single-stranded oligodeoxynucleotides, respectively. As-Cath's wild-type sibling fish, On-target KI of As-Cath, was instrumental in the introduction of the LH knockout catfish line, which featured increased disease resistance and reduced fecundity. In addition, implanting with HCG and LHRH can restore the fecundity, spawnability, and hatchability of the new transgenic fish line's fecundity. Overall, we replaced the LH gene with an alligator cathelicidin transgene and then hormone therapy to achieve complete reproductive control of disease-resistant transgenic catfish in a environmentally sustainable manner.

Source link: https://europepmc.org/article/PPR/PPR592836


CRISPR/Cas9-targeted mutagenesis of TaDCL4, TaDCL5 and TaRDR6 induces male sterility in common wheat.

We discovered RDR6 mutations in plant growth beginning with seed germination and male sterility, growing younger with extensive male sterility, but male sterility was present in dcl5 plants, but not male sterility. In addition, we found that PHAS genes traced by phasiRNAs differed rapidly among subgenomes of polyploid wheat. Genetic dissection of key genes during gametophyte formation can help produce male sterile lines suitable for hybrid wheat breeding, in light of the critical roles of phasiRNA pathways in gametophyte formation.

Source link: https://europepmc.org/article/MED/36597709


A high-efficient and naked-eye visible CRISPR/Cas9 system in Arabidopsis.

Introduction 35S-dsRED2 into the Cas9 vector, which indicates that naked-eye specific dsRED2 greatly enhances the genetic screening, and the WUS promoter driving the Cas9 symbol can increase editing quality in Arabidopsis. In addition, the WUS promoter was used to drive the production of Cas9, which greatly improved the target genes editing efficiency and enabled homozygous mutagenesis of two genes in Arabidopsis' T1 generation. This dsRED2-WUS/Cas9 system could also be used in several crops, considering the conserved function and expression pattern of WUS across the plant species.

Source link: https://europepmc.org/article/MED/36596996


Creation of Two-Line Fragrant Glutinous Hybrid Rice by Editing the Wx and OsBADH2 Genes via the CRISPR/Cas9 System.

Based on meeting the key requirements of high yield and good adaptability, superior eating quality determines the market competitiveness of hybrid rice varieties based on superior eating quality. Cas9, an inexpensive and practical alternative to the lengthy and laborious process of traditional breeding to improve rice quality, is an improved quality for two-line hybrid breeding by editing quality genes with clustered regularly interspaced short palindromic repeat. This paper discusses the use of CRISPR/Cas9 in the production of two-line fragrant glutinous hybrid rice by altering the male sterile and the restorative lines.

Source link: https://europepmc.org/article/MED/36614293


Determining On-Target, Off-Target, and Copy Number Status of Transgenic Events After CRISPR/Cas9 Targeted AAVS1 Safe-Harbor Modification of iPSCs Using Double-Control Quantitative Copy Number PCR.

A recently introduced device that can be used to determine donor DNA insertions in genetically modified monoclonal cell lines is double-control quantitative copy number PCR. The genetic manipulation of immortal cell lines is a powerful way to clarify cell signaling pathways and protein functions. Current techniques to determine the specificity of donor DNA insertions are either expensive or place reliance on manufacturers for assay design and manufacturing. Support Protocol 1: Measurement of monoclonal expansion using immunofluorescence, conversion from plasmid design to monoclonal expansion of reporter protein function; Basic Protocol 1: Genetic transformation at AAVS1 stable harbor in induced pluripotent stem cells using CRISPR/Cas9: From plasmid design to monoclonal expansion using immunofluorescence. PCR Basic Protocol 2: Insert-confirmation PCR Basic Protocol 2: Insert-confirmation PCR reagents and quantification of donor DNA integrations in genetically modified monoclonal cells. Basic Protocol 2: Basic Protocol 2: Introduction and preparation of double-control quantitative copy number PCR reagents and quantification of donor DNA incorporation in genetically modified monoclonal cells.

Source link: https://europepmc.org/article/MED/36598341

* 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

* 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