CRISPR/Cas9 Enhances Organoid Cultivation    

The first intestinal stem cells (Lgr5+) were cultivated into tiny intestine organoids in vitro by Hans Clevers' group in the Netherlands in 2009, marking the beginning of the development of organoid cultivation technology. This discovery established the groundwork for organoid research, which quickly gained popularity as a tool in science. Organoids are in vitro-cultured, three-dimensional miniature organs that closely resemble the composition and capabilities of in vivo tissues. They are extensively employed in drug screening, toxicity testing, illness modeling, genetic development research, and customized treatment regimens.

One of the top biotechnology companies, Creative Biogene, provides organoid gene editing services using state-of-the-art CRISPR/Cas9 technology. This innovative technique broadens the potential applications of organoids in scientific research and clinical trials while improving the precision and efficacy of genetic modifications.

The Power of CRISPR/Cas9 in Organoid Research

CRISPR/Cas9 technology has revolutionized the field of genetic engineering, providing researchers with the tools to make precise modifications to the genome. CRISPR/Cas9 facilitates the creation of genetically edited organoids that closely mimic human tissues and organs when applied to organoid cultivation. These organoids can be used to study complex biological processes, disease mechanisms, and drug responses in a controlled and replicable environment.

Disease models may be produced using CRISPR-Cas9 technology in two major ways: by deleting healthy genes and by repairing damaged genes. First, disease-causing mutations are introduced into healthy cells; second, CRISPR-Cas9 is used to fix mutations in patient-derived cells. This makes it possible to research the causes of diseases and possible cures.

Figure 1. Application CRISPR-Cas9 to generate disease model. (Xiaoshuai L, et al., 2022)

CRISPR-HOT: A Novel Approach for Organoid Gene Editing

The Hubrecht Institute's Hans Clevers team created a brand-new technique called CRISPR-HOT (CRISPR/Cas9-mediated Homology-Independent Organoid Transgenesis) in March 2020. For gene knock-in, this technique employs non-homologous end joining (NHEJ). When it comes to introducing transgenes into the genome, CRISPR-HOT offers great flexibility and efficiency because homologous recombination is not required. Exogenous DNA may be inserted into a variety of organoid models, such as those made from the intestines, liver, and other tissues, using CRISPR-HOT. This approach streamlines the genome editing procedure and offers a dependable way to see certain genes in human organoids. This approach was used to analyze the division of liver cells and the emergence of aberrant liver cells that are rich in DNA. Knocking out the cancer gene TP53 led to more frequent unstructured divisions of abnormal liver cells, potentially revealing processes of cancer development.

CRISPR-HOT Versus Traditional Methods

A major improvement over conventional homology-directed repair (HDR) techniques is CRISPR-HOT. Clevers' group showed that NHEJ-mediated CRISPR-HOT produced noticeably greater gene insertion efficiency in a comparison analysis. They found that NHEJ-mediated insertions were more common than HDR-mediated insertions (0.9–0.7% efficiency for HDR vs. 9%–14% for NHEJ) using the NEPA21 high-efficiency gene transfection method. This significant advancement highlights CRISPR-HOT's promise for a range of organoid models.

Technical Overview

Custom sgRNA Design and Cloning

The organoid gene editing service starts with creating and cloning single guide RNAs (sgRNAs) that are unique to target genes. The endogenous loci that our specialists target with sgRNAs are ideally in the last exon and around the stop codon. To find the best sequence for your research objectives, many sgRNAs are evaluated.

Preparation of DNA Constructs

We make ready the DNA constructs required for transfection, such as the universal NHEJ targeting vector, the appropriate frame selector plasmid, and the sgRNA plasmid that targets the locus of interest. This guarantees that the elements needed for effective gene editing are easily accessible and well-suited for organoid transfection.

Organoid Transfection Protocols

Protocols for transfection are tailored to the specific type of organoid. For small intestine organoids, liver ductal organoids, and hepatocyte organoids, for example, we adhere to known techniques while adding particular modifications to guarantee maximum gene editing effectiveness. In order to enable high-efficiency gene integration, our team introduces the DNA constructs into the organoids using sophisticated electroporation methods.

Selection and Expansion of Knocked-In Organoids

We use fluorescence-based selection after transfection to determine which altered organoids are effective. Fluorescent marker-tagged organoids are separated and grown. After that, in order to verify that the intended genetic alterations have been correctly integrated, we do genotyping using Sanger sequencing.

Troubleshooting and Optimization

We provide comprehensive troubleshooting and optimization throughout the gene editing process. If fluorescence is absent, we analyze gene expression and conduct experiments with alternative sgRNAs or gene regions. We also suggest strategies to improve selection, such as FAC-sorting or using resistance genes with separate promoters.

Creative Biogene's enhanced organoid culture service, powered by CRISPR/Cas9, offers researchers a potent tool for precise genetic alterations. Our ability to create intricate organoid models is vital for advancing biomedical research and drug development. Count on us for dependable, high-quality results tailored to your specific scientific needs. Visit our website or contact our specialist team for more information on how our organoid gene editing services can benefit your research.

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