Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Liu, Kaiwen Ivy; Sutrisnoh, Norfala-Aliah Binte; Wang, Yuanming; Tan, Meng How

doi:10.3791/59086

Cited by 5 publications

(4 citation statements)

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“…After inducing the CRISPR (Cas9) nuclease expression associated in cells transfected by the viral vector, the nuclease forms a complex with the guide RNA leading to DNA cleavage in the specific target sequence directed by the guide RNA right after a PAM motif (protospacer adapter motif). Cas9 cleavage promotes double-strand breaks, activating DNA repair mechanisms by Non-Homologous End Joining (NHEJ), promoting the mutation of the target site, with consequent gene inactivation (Gao et al, 2020;Liu et al, 2019).…”

Section: Viral Vectorsmentioning

confidence: 99%

Cannabidiol as an add-on therapy to overcome the slow-onset and, possibly, resistance to antidepressant treatment: involvement of NAPE-PLD in the medial prefrontal cortex

Ff¹,

Ej³

et al. 2021

Preprint

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Antidepressants such as serotonin uptake inhibitors are the first-line pharmacological treatment for chronic stress-related psychiatric disorders. However, their late-onset therapeutic action and frequent side effects, however, are important challenges for clinicians and patients. Besides, around 30% of major depression patients are considered treatment-resistant. Cannabidiol (CBD) is a non-psychotomimetic phytocannabinoid with a wide range of psychopharmacological effects, but its mechanism of action remains unclear. Here, we found that in male mice submitted to two different repeated stress protocols (chronic unpredictable and social defeat stress), low doses of CBD (7.5mg/Kg) caused an early-onset behavioral effect when combined to the antidepressant escitalopram (ESC-10mg/Kg). The behavioral effects of the ESC+CBD combination depended on the expression/activity of the N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD, responsible for synthesizing the endocannabinoid anandamide), but not the DAGLα, enzyme in the ventromedial prefrontal cortex. In addition, we described a case series with three treatment-resistant depression that were successfully treated with CBD as adjuvant therapy, as evaluated by standardized clinical rating scales. After 12 weeks of treatment, two patients were considered depression remitted (MADRS score lower than 10) while one patient successfully responded to CBD as add-on treatment (more than 50% decrease from the baseline MADRS). Our results suggest that CBD might be useful as an add-on therapy for optimizing the action of antidepressants. They also suggest that CBD’s beneficial actions depends on the facilitation of N-acylethanolamines actions in the medial prefrontal cortex.HighlightsIn mice, cannabidiol (CBD), but not escitalopram, induced a fast-onset anti-stress action.Combinations of sub-effective doses of CBD and escitalopram produce anti-stress effects after only 7 days.The Escitalopram + CBD treatment modulated synaptic protein markers in the medial prefrontal cortex.CRISPR-Cas9-mediated knockdown of NAPE-PLD in the medial PFC prevents the anti-stress effect of the Escitalopram + CBD.Adding CBD to an antidepressants regimen successfully treated three patients with treatment resistant depression.Graphical abstract

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Section: Viral Vectorsmentioning

confidence: 99%

Cannabidiol as an add-on therapy to overcome the slow-onset and, possibly, resistance to antidepressant treatment: involvement of NAPE-PLD in the medial prefrontal cortex

Ff¹,

Ej³

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…In recent years, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has emerged as a powerful approach to edit the mammalian genome, including modulation of a target gene's expression [17,18]. Although this newer technology has been used less extensively than RNAi to investigate integrins, CRISPR-mediated ablation of integrin expression has been applied in different contexts [19][20][21][22][23][24][25].…”

Section: Introductionmentioning

confidence: 99%

Comparative use of CRISPR and RNAi to modulate integrin α3β1 in triple negative breast cancer cells reveals that some pro-invasive/pro-metastatic α3β1 functions are independent of global regulation of the transcriptome

et al. 2021

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Integrin receptors for the extracellular matrix play critical roles at all stages of carcinogenesis, including tumor growth, tumor progression and metastasis. The laminin-binding integrin α3β1 is expressed in all epithelial tissues where it has important roles in cell survival, migration, proliferation, and gene expression programs during normal and pathological tissue remodeling. α3β1 signaling and adhesion functions promote tumor growth and metastasis in a number of different types of cancer cells. Previously, we used RNA interference (RNAi) technology to suppress the expression of the ITGA3 gene (encoding the α3 subunit) in the triple-negative breast cancer cell line, MDA-MB-231, thereby generating variants of this line with reduced expression of integrin α3β1. This approach revealed that α3β1 promotes pro-tumorigenic functions such as cell invasion, lung metastasis, and gene regulation. In the current study, we used CRISPR technology to knock out the ITGA3 gene in MDA-MB-231 cells, thereby ablating expression of integrin α3β1 entirely. RNA-seq analysis revealed that while the global transcriptome was altered substantially by RNAi-mediated suppression of α3β1, it was largely unaffected following CRISPR-mediated ablation of α3β1. Moreover, restoring α3β1 to the latter cells through inducible expression of α3 cDNA failed to alter gene expression substantially, suggesting that use of CRISPR to abolish α3β1 led to a decoupling of the integrin from its ability to regulate the transcriptome. Interestingly, both cell invasion in vitro and metastatic colonization in vivo were reduced when α3β1 was abolished using CRISPR, as we observed previously using RNAi to suppress α3β1. Taken together, our results show that pro-invasive/pro-metastatic roles for α3β1 are not dependent on its ability to regulate the transcriptome. Moreover, our finding that use of RNAi versus CRISPR to target α3β1 produced distinct effects on gene expression underlines the importance of using multiple approaches to obtain a complete picture of an integrin’s functions in cancer cells.

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“…CRISPR/Cas9 technology is used in a wide range areas suchlike identification of bacterial strains, determination of gene and miRNA functions, addition/deletion of DNA fragments to the genome, transcriptional and epigenetic targeting or generation of disease models (Rodriguez et al, 2019;Li et al, 2020). CRISPR/Cas9 technology has started a new era in genome editing with its feasibility in mammalian cells (Liu et al, 2019). Today, scientists can easily edit genomes, using CRISPR/Cas9mediated tools (Hsu et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

The Effect Of Molecular Genetic Mechanisms On Drug Addiction And Related New Generation CRISPR Gene Engineering Applications

Akyürek¹,

Uysal²,

Gulden³

et al. 2021

genediting

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Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Cited by 5 publications

References 0 publications

Cannabidiol as an add-on therapy to overcome the slow-onset and, possibly, resistance to antidepressant treatment: involvement of NAPE-PLD in the medial prefrontal cortex

Cannabidiol as an add-on therapy to overcome the slow-onset and, possibly, resistance to antidepressant treatment: involvement of NAPE-PLD in the medial prefrontal cortex

Comparative use of CRISPR and RNAi to modulate integrin α3β1 in triple negative breast cancer cells reveals that some pro-invasive/pro-metastatic α3β1 functions are independent of global regulation of the transcriptome

The Effect Of Molecular Genetic Mechanisms On Drug Addiction And Related New Generation CRISPR Gene Engineering Applications

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