Dataset Information

DIPA-CRISPR is a simple and accessible method for insect gene editing.

ABSTRACT: Current approaches for insect gene editing require microinjection of materials into early embryos. This severely limits the application of gene editing to a great number of insect species, especially to those whose reproduction systems preclude access to early embryos for injection. To overcome these limitations, we report a simple and accessible method for insect gene editing, termed "direct parental" CRISPR (DIPA-CRISPR). We show that injection of Cas9 ribonucleoproteins (RNPs) into the haemocoel of adult females efficiently introduces heritable mutations in developing oocytes. Importantly, commercially available standard Cas9 protein can be directly used for DIPA-CRISPR, which makes this approach highly practical and feasible. DIPA-CRISPR enables highly efficient gene editing in the cockroaches, on which conventional approaches cannot be applied, and in the model beetle Tribolium castaneum. Due to its simplicity and accessibility, DIPA-CRISPR will greatly extend the application of gene editing technology to a wide variety of insects.

SUBMITTER: Shirai Y

PROVIDER: S-EPMC9142683 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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DIPA-CRISPR is a simple and accessible method for insect gene editing.

Shirai Yu Y Piulachs Maria-Dolors MD Belles Xavier X Daimon Takaaki T

Cell reports methods 20220516 5

Current approaches for insect gene editing require microinjection of materials into early embryos. This severely limits the application of gene editing to a great number of insect species, especially to those whose reproduction systems preclude access to early embryos for injection. To overcome these limitations, we report a simple and accessible method for insect gene editing, termed "direct parental" CRISPR (DIPA-CRISPR). We show that injection of Cas9 ribonucleoproteins (RNPs) into the haemoc ...[more]

PMID: 35637909

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Project description:BackgroundCRISPR-Cas9-based technologies have revolutionized experimental manipulation of mammalian genomes. None-the-less, limitations of the delivery and efficacy of these technologies restrict their application in primary cells.AimsTo create an optimized protocol for penetrant, reproducible, and fast targeted genome editing in cell cultures derived from primary cells, using patient-derived glioblastoma stem-like cells (GSCs) and human neural stem/progenitor cells (NSCs) for proof-of-concept experiments.Methods and resultsWe employed transient nucleofection of Cas9:sgRNA ribonucleoprotein complexes composed of chemically synthesized 2'-O-methyl 3'phosphorothioate-modified sgRNAs and purified Cas9 protein. Insertion-deletion mutation (indel) frequency and size distribution were measured via computational deconvolution of Sanger sequencing trace data. We found that this optimized technique routinely allows for >90% indel formation in only 3 days, without the need to create clonal lines for simple loss-of-function experiments. Using Western blotting, we observed near-total protein loss of target genes in cell pools. Additionally, we found that this approach allows for the creation of targeted genomic deletions. Furthermore, by using RNA-seq in edited NSCs to assess gene expression changes resulting from knockout of tumor suppressors commonly altered in glioblastoma, we also demonstrated the utility of this method for quickly creating a series of gene knockouts that allow for the study of oncogenic activities.ConclusionOur data suggest that this relatively simple method can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells (such as GSCs). This represents an extremely useful tool for the cancer research community when wishing to inactivate not only coding genes, but also non-coding RNAs, UTRs, enhancers, and promoters. This method can be readily applied to diverse cell types by varying the nucleofection conditions.

Project description:Background: CRISPR-Cas9-based technologies have revolutionized experimental manipulation of mammalian genomes. None-the-less, limitations of the delivery and efficacy of these technologies restrict their application in primary cells. Aims: To create an optimized protocol for penetrant, reproducible, and fast targeted genome editing in cell cultures derived from primary cells, using patient-derived glioblastoma (GBM) stem-like cells (GSCs) and human neural stem/progenitor cells (NSCs) for proof-of-concept experiments. Methods and results: We employed transient nucleofection of Cas9:sgRNA ribonucleoprotein complexes composed of chemically synthesized 2'-O-methyl 3’phosphorothioate-modified sgRNAs and purified Cas9 protein. Insertion-deletion mutation (indel) frequency and size distribution were measured via computational deconvolution of Sanger sequencing trace data. We found that this optimized technique routinely allows for >90% indel formation in only 3 days, without the need to create clonal lines for simple loss-of-function experiments. Using Western blotting, we observed near-total protein loss of target genes in cell pools. Additionally, we found that this approach allows for the creation of targeted genomic deletions. By using RNA-seq in edited NSCs to assess gene expression changes resulting from knockout of tumor suppressors commonly altered in GBM, we also demonstrated the utility of this method for quickly creating a series of gene knockouts that allow for the study of oncogenic activities. Conclusion: Our data suggest that this relatively simple method can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells (such as GSCs). This represents an extremely useful tool for the cancer research community when wishing to inactivate not only coding genes, but also non-coding RNAs, UTRs, enhancers, and promoters. This method can be readily applied to diverse cell types by varying the nucleofection conditions.

Dataset Information

DIPA-CRISPR is a simple and accessible method for insect gene editing.

Publications

DIPA-CRISPR is a simple and accessible method for insect gene editing.

Similar Datasets

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