New Trends in Gene Editing - Knockout Cells and Prime Editing Technology Illuminate the Road to Genetic Research

K nockout cells decipher the mechanism of cell antiviral ability

Guanylate-binding prot eins (GBPs) are highly expressed interferon-stimulated genes (ISGs) that have antagonistic effects on various intracellular pathogenic microorganisms. ECTV has very similar genetic and disease characteristics to monkeypox virus, making it a valuable research tool for studying orthopoxvirus-host interactions. Guanylate-binding proteins (GBPs) are highly expressed interferon-stimulated genes (ISGs) that have antagonistic effects on various intracellular pathogenic microorganisms.

The gRNA plasmid containing the specific targeting gene PAPL was transfected into small hamster kidney cells (BRT17 cells), and then the small hamster kidney cells were infected by ECTV, and subsequently obtained the PAPL knockout ECTV. Viral plaque assays were performed in African green monkey kidney fibroblasts (CV1) to evaluate the effect of PAPL deletion on viral replication. The replication ability of the virus in cells lacking the PAPL gene was significantly reduced, indicating that PAPL plays an indispensable role in the replication process of ECTV. More details>>

Figure 1. PAPL gene knockout results in reduced virus replication in vitro

M ulti-model study on new PKC isoforms in DNA integrity checkpoints by PKCδ knockout cells

The protein kinase C (PKC) family plays an important regulator in many cellular metabolic processes. Both Pkc1 and the new isoform PKCδ are involved in the regulation of DNA integrity checkpoint activation, and this mechanism is conserved from yeast to mammals .

PKCδ knockout mouse embryonic stem cells (mESCs) were obtained using CRISPR-Cas9 technology. This model suggests that loss of PKCδ reduces activation of the effector kinase CHK1, however other isoforms may be involved in this function. Therefore, the researchers used yeast to study the ability of each single PKC isoform to deactivate the DNA integrity checkpoint. The researchers' analysis found that PKCθ, the isoform closest to PKCδ, is also able to perform this function, through it is less efficient. This study validates the critical role of PKC in the DNA integrity checkpoint pathway and emphasize s the advantages of combining different research models.

Figure 2. Analysis of the role of PKCδ in activation of DNA integrity checkpoint in mouse ESCs

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Using prime editing technology to obtain FecBB and TBXT mutant sheep and KCNJ5G151R/+ point mutant pig blastocyst

The recent emergence of prime editing (PEs) that can achieve all possible single nucleotide substitutions as well as targeted insertions and deletions has shown promising potential for editing and correcting the genomes of various organisms. However, studies on the application of PE in editing livestock genomes have not yet been reported.

In this study, researchers used PE technology to successfully generate two mutant sheep for increasing agricultural production, including FecBBp, which is related to fertility. Q249R and tail length related TBXT p.G112W. In addition, the researchers applied PE technology to produce a porcine blastocyst (KCNJ5 p.G151R) with a biomedically relevant point mutation as a porcine model of human primary aldosteronism. The application of this study demonstrates the potential of the Prime editing large animal genomes and provides an important microcosm of the recent emergence of major editing systems to achieve livestock genome mutations in agriculture and biomedicine. More details>>