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The new research was done to determine the effectiveness of genome editing in diploid and tetraploid potato varieties. The stem internodes of AGB and M6 plants were infected with Agrobacterium strain LBA4404, carrying the CRISPR/Cas9 virus, which carried a specific guide RNA against vacuolar invertase and Cas9 endonuclease. In comparison to the tetraploid variety that rooted without IBA, the regenerated plantlets were transferred to culture tubes, where the M6 diploid variety needed indole-3-butyric acid for root growth. Overall, the findings revealed that the adopted transformation protocol returned plants with edited target genes that can be used in a more effective potato breeding program.
Source link: https://doi.org/10.1007/s11738-022-03513-4
Introduction 35S - dsRED2 into the Cas9 vector, which implies naked-eye readable dsRED2 results in genetic testing, and the WUS promoter driving the Cas9 expression can enhance editing quality in Arabidopsis. In addition, the WUS promoter was used to stimulate Cas9's gene expression, which also enabled homozygous mutagenesis of two genes in Arabidopsis' T1 generation. Given the preserved function and expression pattern of WUS across the plant species, this dsRED2-WUS/Cas9 system could also be used in several crops.
Source link: https://doi.org/10.1007/s00425-022-04060-5
Although several enzymes of the PARP family could synthesize it in all multicellular organisms, Drosophila melanogaster is a particularly good model for studying pADPr-regulated processes because only one PARP gene is present. Despite the fact that PARP is an extensively researched protein with multiple important functions, no complete knockout PARP flies were found in mobile element mutagenesis-based studies, mainly because PARP gene localizes in a heterochromatic region. Here, we describe all steps of creating PARP mutated D. melanogaster with CRISPR/Cas9 technology from the gRNA blueprint, plasmid cloning to fly crosses, and mutation detection.
Source link: https://doi.org/10.1007/978-1-0716-2891-1_21
However, some tumor cell lines and primary tumors, as well as some tumor cell lines, indicate resistance to therapy. Here we introduce an approach to determining novel host factors responsible for tumor resistance to oncolysis by a non-function screening that uses an objective genome-wide CRISPR-Cas9 loss-of-function analysis. Cas9-expressing tumor cells are transduced with a pooled single-guide RNA expressing lentiviruses that target all human genes in order to establish a cell population where each cell is knocked out for a single gene by a single gene. Resistent cells survive after OV infection, although sensitive cells die.
Source link: https://doi.org/10.1007/978-1-0716-2788-4_25
Although functional genetic methods have long been established in a variety of standard model systems, genetic mutations have historically been present in organisms used in evolutionary studies. Gene expression in A. mexicous is described here. Using CRISPR-Cas9 in A. mexicanus, we want to provide guidelines for performing functional genetic tests and to highlight the benefits of using this technique.
Source link: https://doi.org/10.1007/978-1-0716-2875-1_14
Duchenne muscular dystrophy is a fatal neuromuscular disorder attributed to DMD gene coding dystrophin mutations. In particular, CRISPR-Cas9 gene editing kits included by self-complementary AAV, enabling the restoration of dystrophin expression in animal models of DMD. Here, we describe protocols for cloning CRISPR single guide RNAs from a scAAV plasmid and methods for systemic delivery of AAVs into a DMD mouse model.
Source link: https://doi.org/10.1007/978-1-0716-2772-3_21
In fact, AAV vectors have been used to produce genomic exon skipping and restore the dystrophin protein in several Duchenne muscular dystrophy model animals. Despite the high transduction rate, AAV vector-mediated delivery suffers several drawbacks, including the packaging size, prolonged overexpression of Cas9, immunogenicity against the AAV capsid, and the danger of inserting a portion of the AAV genome sequence into the host cell. NanoMEDIC converts Cas9 protein and gRNA into EVs by an HIV-derived Gag protein. The Cas9 and Gag proteins are fused to a heterodimerizer and conditionally dimerized by the addition of an inducible chemical ligand to convert Cas9 protein into EVs. NanoMEDIC can produce highly accurate packaging of the Cas9 protein and gRNA for genome editing into a variety of target cells and in vivo by using these attributes.
Source link: https://doi.org/10.1007/978-1-0716-2772-3_22
Phages are viruses of bacteria that have been around for more than a century, and have been known for over a century. The following information explains how a CRISPR-Cas9 device with an artificial CRISPR-array can be constructed and used to distinguish abundant and well-known B. subtilis phage enrichment from a host-based metagenome enrichment. The resulting viral suspension can be used for metagenome sequencing and isolating new viral strains.
Source link: https://doi.org/10.1007/978-1-0716-2795-2_15
A progressive myopathy exacerbated by mutations in genes encoding dystrophin proteins that eventually result in the depletion of myogenic progenitor cells, which eventually led to the depletion of myogenic progenitor cells. Several strategies have been used to correctly express the dystrophin gene in induced pluripotent stem cells, including deletion of mutated exon 23 by clustered short palindromic repeat/CRISPR-associated gene editing technology.
Source link: https://doi.org/10.1007/978-1-0716-2772-3_23
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