CRISPR/Cas9 Gene Editing System Can Alter Gene Expression and Induce DNA Damage Accumulation

Yang, Lan; Li, Hao; Han, Yao; Song, Yingjie; Wei, Mingchen; Fang, Mengya; Sun, Yuliang

doi:10.3390/genes14040806

Cited by 4 publications

(3 citation statements)

References 29 publications

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“…Editing of NFYA gene resulted in 32% decrease of mRNA and 66.6% decrease of protein. Previous finding found that changes in the mRNA levels after gene editing are positively correlated with the gene editing efficiency [14]. Several manipulations of mRNA after CRISPR/Cas9 were documented.…”

Section: Discussionmentioning

confidence: 93%

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CRISPR/Cas9 alter NFYA binding site on CD44-regulating cis-element and control CD44 expression in breast cancer cells

Salem,

Yahya

2024

Preprint

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Background CRISPR/Cas9 is used for editing of non-coding sequence. This study tested the involvement of downstream cis-element in regulating CD44 expression and the possibility of using CRISPR/Cas9 system to manipulate the transcription factor binding site. Bioinformatic tools predicted two binding sites (P1 and P2) for NFYA transcription factors. CRISPR/Cas9 was used to knockout NFYA gene and alter the NFYA binding site (P2). Results The data revealed decrease of CD44 gene expression and CD44+ CD24− sub-population after editing of P2 sequence more than the decrease resulted from editing of NFYA gene itself, confirming the involvement of NFYA in regulating CD44 gene. Both editing inhibited the migration ability of MDA-MB-231 cells. Unlike editing of NFYA gene, altering P2 sequence induced apoptosis. CHIP assay revealed that NFY have the ability to bind both P1 and P2 sequences, with higher enrichment in case of P2 than P1. After performing site-directed mutagenesis and luciferase assay we confirmed the involvement of both P1 and P2 in gene regulation, with higher potential in case of P2 than P1. Conclusion The findings confirmed the regulation of CD44 by NFYA and the efficacy of using CRISPR/Cas9 in altering the binding site, and downregulation of CD44.

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Section: Discussionmentioning

confidence: 93%

“…Ten exons (1-5 and 16-20) produce the standard form of CD44 (CD44s; ~85 kDa) and known as "constant" exons. The ten central exons (6)(7)(8)(9)(10)(11)(12)(13)(14)(15) subjected to extensive alternative splicing to form splicing variants (CD44v isoforms) (CD44v) and known as variant exons 1-10 (v1-10) [5,6].…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9 alter NFYA binding site on CD44-regulating cis-element and control CD44 expression in breast cancer cells

Salem,

Yahya

2024

Preprint

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“…73 The above could be explained because during the active period of gene editing, changes occur in chromosome segregation and nuclear division, evidencing a probable mechanism for the induction of chromosomal alterations following gene editing. 88 Overall, the research carried out to date shows a limitation for the application of DSB-inducing CRISPR therapy in the clinic, so it is important to consider the possibility that extensive chromosomal rearrangements may be induced as a result of the application of this gene editing technology.…”

Section: Crispr-cas9 Limitations-induction Of Chromosomal Alterationsmentioning

confidence: 99%

Exploring the Advantages and Limitations of CRISPR-Cas in Breast Cancer

Rangel,

Camargo,

Castellanos

et al. 2024

Gene Expr

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Breast cancer (BC) is the type of cancer with the highest incidence and mortality rates in women in the world. In the treatment of this neoplasia, several therapies are applied, including radiotherapy, hormonal therapy, chemotherapy, and biological therapy. Although most patients respond to these types of therapy, some patients over time, develop resistance or eventually relapse. Considering the above, future therapeutic concepts in BC are being directed at individualization of therapy and escalation of treatment based on tumor biology through the use of gene therapy. In this regard, a new genomic engineering technology, called the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein-9 (Cas9), has acquired great importance in recent years, as a potential gene editing tool, extensively applied in human cancer research and cancer treatment. The aim of this review was to describe the advantages, limitations, and applications of CRISPR gene editing technology in BC treatment. Our review emphasizes the innovative facets and profound importance of CRISPR gene editing technology within the BC treatment landscape. Additionally, it provides valuable information to consider when evaluating the risks associated with the implementation of CRISPR-Cas9 technology in BC therapy.

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Generation and Characterization of a NovelPrkcd-Cre Rat Model

Toivainen,

Petrella,

et al. 2024

J. Neurosci.

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Activity of central amygdala (CeA) PKCδ expressing neurons has been linked to appetite regulation, anxiety-like behaviors, pain sensitivity, and addiction-related behaviors. Studies of the role that CeA PKCδ+ neurons play in these behaviors have largely been carried out in mice, and genetic tools that would allow selective manipulation of PKCδ+ cells in rats have been lacking. Here, we used a CRISPR/Cas9 strategy to generate a transgenicPrkcd-cre knock-in rat, and characterized this model using anatomical, electrophysiological and behavioral approaches in both sexes. In the CeA, Cre was selectively expressed in PKCδ+ cells. Anterograde projections of PKCδ+ neurons to cortical regions, subcortical regions, several hypothalamic nuclei, the amygdala complex, and midbrain dopaminergic regions were largely consistent with published mouse data. In a behavioral screen, we found no differences between Cre+rats and Cre-wildtype littermates. Optogenetic stimulation of CeA PKCδ+ neurons in a palatable food intake assay resulted in an increased latency to first feeding and decreased total food intake, once again replicating published mouse findings. Lastly, using a real-time place preference task, we found that stimulation of PKCδ+ neurons promoted aversion, without affecting locomotor activity. Collectively, these findings establish the novelPrkcd-Cre rat line as a valuable tool, that complements available mouse lines for investigating the functional role of PKCδ+ neurons.Significance StatementThe central nucleus of the amygdala (CeA), involved in processing threat and aversion signals, comprises multiple neuronal subtypes. Expression of protein kinase C isoform δ, PKCδ, marks CeA neurons that respond to aversive stimuli, and have also been shown to play a role in alcohol-related behaviors. Genetic tools to investigate the functional role of PKCδ+ neurons in rat models have been lacking. We describe the development and characterization of a novelPrkcdknock-in transgenic rat generated using CRISPR strategy. In this model, we confirm known projection targets of CeA PKCδ+ neurons and replicate functional consequences of their activation previously found in mice. This establishes the line as a novel model to study the role of PKCδ+ neurons in rat models.

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CRISPR/Cas9 Gene Editing System Can Alter Gene Expression and Induce DNA Damage Accumulation

Cited by 4 publications

References 29 publications

CRISPR/Cas9 alter NFYA binding site on CD44-regulating cis-element and control CD44 expression in breast cancer cells

CRISPR/Cas9 alter NFYA binding site on CD44-regulating cis-element and control CD44 expression in breast cancer cells

Exploring the Advantages and Limitations of CRISPR-Cas in Breast Cancer

Generation and Characterization of a NovelPrkcd-Cre Rat Model

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