Efficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPR
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ABSTRACT:
PROVIDER: PRJNA1009059 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR25757213_1.fastq.gz | Fastqsanger.gz | |||
| SRR25757213_2.fastq.gz | Fastqsanger.gz | |||
| SRR25757214_1.fastq.gz | Fastqsanger.gz | |||
| SRR25757214_2.fastq.gz | Fastqsanger.gz | |||
| SRR25757215_1.fastq.gz | Fastqsanger.gz |
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Project description:Organoids are currently one of the most widely used ex vivo models in epithelial biology. Combined with genetic editing strategies, organoids offer a promise of rapid and efficient investigation of gene function in many models of human disease. However, to date, the editing efficiency of organoids with the use of non-viral electroporation methods has been only up to 30%, with implications for the subsequent need for selection including turnaround time and exhaustion or adaptation of the organoid population. Here, we describe an efficient method of intestinal organoid editing using a Ribonucleoprotein CRISPR-based approach. Editing efficiencies of up to 98% in target genes were robustly achieved across different anatomical gut locations and developmental timepoints from multiple patient samples with no observed off-target editing. The method allowed us to study the effect of the loss of the tumour suppressor gene, PTEN, in normal human intestinal cells. Analysis of PTEN deficient organoids defined phenotypes that likely relate to its tumour suppressive function in vivo, such as a proliferative advantage and increased organoid budding. Transcriptional profiling revealed differential expression of genes in pathways commonly known to be associated with PTEN loss including mTORC1 activation.
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Project description:The CRISPR-Cas9 system enables efficient sequence-specific mutagenesis for creating germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we established in vitro-assembled, fluorescent Cas9-sgRNA RNPs in stabilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. Sequence analysis of targeted loci in individual embryos reveals highly efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis reveals preliminary loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show efficient targeting of functional non-coding elements in gene-regulatory regions using saturating mutagenesis towards uncovering functional control elements in transgenic reporters and endogenous genes. Our results suggest that in vitro assembled, fluorescent Cas9-sgRNA RNPs provide a rapid reverse-genetics tool for direct and scalable loss-of-function studies beyond zebrafish applications.
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Project description:We evaluate CRISPR-based prime editing for application in organoids. First we model mutations in TP53 in intestinal and hepatocyte oganoids and determine the efficiency and accuracy of mutation induction on multiple targets. Then, to evaluate potential clinical applicability of prime editing we repair mutations in the CFTR channel that cause cystic fibrosis in intestinal organoids. First we repair the CFTR-F508del mutation which is the most common mutation in cystic fibrosis. Then we compare adenine base editing to prime editing by repairing the CFTR-R785* mutation using both strategies.
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Project description:Genome editing through the delivery of CRISPR/Cas9-ribonucleoprotein (Cas9-RNP) reduces unwanted gene targeting and avoids integrational mutagenesis that can occur through gene delivery strategies. Direct and efficient delivery of Cas9-RNP into the cytosol followed by translocation to the nucleus remains a challenge. Here, we report a remarkably highly efficient (∼90%) direct cytoplasmic/nuclear delivery of Cas9 protein complexed with a guide RNA (sgRNA) through the coengineering of Cas9 protein and carrier nanoparticles. This construct provides effective (∼30%) gene editing efficiency and opens up opportunities in studying genome dynamics.
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Project description:The CRISPR/Cas9 system has been employed to efficiently edit the genomes of diverse model organisms. CRISPR-mediated mouse genome editing is typically accomplished by microinjection of Cas9 DNA/RNA and single guide RNA (sgRNA) into zygotes to generate modified animals in one step. However, microinjection is a technically demanding, labor-intensive, and costly procedure with poor embryo viability. Here, we describe a simple and economic electroporation-based strategy to deliver Cas9/sgRNA ribonucleoproteins into mouse zygotes with 100% efficiency for in vivo genome editing. Our methodology, designated as CRISPR RNP Electroporation of Zygotes (CRISPR-EZ), enables highly efficient and high-throughput genome editing in vivo, with a significant improvement in embryo viability compared with microinjection. Using CRISPR-EZ, we generated a variety of editing schemes in mouse embryos, including indel (insertion/deletion) mutations, point mutations, large deletions, and small insertions. In a proof-of-principle experiment, we used CRISPR-EZ to target the tyrosinase (Tyr) gene, achieving 88% bi-allelic editing and 42% homology-directed repair-mediated precise sequence modification in live mice. Taken together, CRISPR-EZ is simple, economic, high throughput, and highly efficient with the potential to replace microinjection for in vivo genome editing in mice and possibly in other mammals.
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Project description:The Fusarium oxysporum species complex (FOSC) is an economically important group of pathogenic filamentous fungi that are able to infect both animals and plants. Reverse genetic techniques, including gene disruption/deletion methods, to study these fungi are available although limitations exist resulting in decreased efficiency. Herein we describe a gene editing system developed using a F. oxysporum-optimized Cas9 ribonucleoprotein (RNP) and protoplast transformation method. The Cas9 protein and sgRNA were assembled to form a stable RNP in vitro and this complex was transferred into fungal protoplasts for gene editing with PEG-mediated transformation. In order to determine if the Cas9 RNP system is functional in the FOSC protoplasts and assess the efficacy of the system, two genes, URA5 and URA3, were selected for targeted disruption generating uracil auxotroph mutants that are resistant to 5-fluoroorotic acid, 5-FOA. In addition, a gene in a secondary metabolite biosynthetic cluster, the ortholog of BIK1, was mutated using this system and the maximum efficiency of this gene disruption was about 50%. Further analysis of the bik1 mutant confirmed that this polyketide synthase was involved in the synthesis of the red pigment, bikaverin. The mutants generated in this study displayed the strong expected phenotypes, demonstrating this F. oxysporum-optimized CRISPR/Cas9 system is stable and can efficiently disrupt the genes of interest.
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Project description:Gene editing with CRISPR/Cas9 is a powerful tool to study the function of target genes. Although this technology has demonstrated wide efficiency in many species, including fertilized zebrafish and medaka fish embryos when microinjected, its application to achieve efficient gene editing in cultured fish cells have met some difficulty. Here, we report an efficient and reliable approach to edit genes in cultured medaka (Oryzias latipes) fish cells using pre-formed gRNA-Cas9 ribonucleoprotein (RNP) complex. Both medaka fish haploid and diploid cells were transfected with the RNP complex by electroporation. Efficient gene editing was demonstrated by polymerase chain reaction (PCR) amplification of the target gene from genomic DNA and heteroduplex mobility assay carried out with polyacrylamide gel electrophoresis (PAGE). The heteroduplex bands caused by RNP cleavage and non-homologous end joining could be readily detected by PAGE. DNA sequencing confirmed that these heteroduplex bands contains the mutated target gene sequence. The average gene editing efficiency in haploid cells reached 50%, enabling us to generate a clonal cell line with ntrk3b gene mutation for further study. This RNP transfection method also works efficiently in diploid medaka cells, with the highest mutation efficiency of 61.5%. The specificity of this synthetic RNP CRISPR/Cas9 approach was verified by candidate off-target gene sequencing. Our result indicated that transfection of pre-formed gRNA-Cas9 RNP into fish cells is efficient and reliable to edit target genes in cultured medaka fish cells. This method will be very useful for gene function studies using cultured fish cells.
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Project description:Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA. Consistent with this finding, no off-target mutations are detected in the mutant plants. Because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing, the mutants obtained are completely transgene free. This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized.
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Project description:Genome editing has transformative potential for curing human diseases but poses unique challenges due to irreversible DNA alterations. Ensuring safe and effective in vivo editing requires preclinical studies to mitigate risks like genotoxicity, cytotoxicity, and immunogenicity. The kidney, highly susceptible to off-target effects due to its blood flow and exposure to systemic therapies, lacks established human models for assessing gene therapies. We utilized kidney organoids as a preclinical platform to test CRISPR/Cas9 genome editing via adeno-associated virus (AAV) delivery, a clinical strategy raising safety concerns due to toxic viral titers. We conduced RNA-seq to assess nephrotoxic responses.
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