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Related Concept Videos

CRISPR01:59

CRISPR

50.9K
Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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CRISPR and crRNAs02:53

CRISPR and crRNAs

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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
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Related Experiment Video

Updated: Jun 30, 2025

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins
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Optimizing CRISPR/Cas9 approaches in the polymorphic tunicate Ciona intestinalis.

Alessandro Pennati1, Miloš Jakobi1, Fan Zeng1

  • 1Institute of Zoology, University of Innsbruck, 6020, Innsbruck, Austria; Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, 6020, Innsbruck, Austria.

Developmental Biology
|March 15, 2024
PubMed
Summary
This summary is machine-generated.

We optimized CRISPR/Cas9 gene editing in the Ciona intestinalis model organism by improving guide RNA design, validating in vitro assays, and enhancing in vivo delivery. These advancements increase CRISPR knockout efficiency and are transferable to other species.

Keywords:
CRISPRCas9:GemininCiona intestinalisPolymorphismTunicates

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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

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Area of Science:

  • * Developmental Biology
  • * Molecular Biology
  • * Genetics

Background:

  • * Ciona intestinalis is a key invertebrate model for vertebrate developmental studies due to shared features and experimental advantages.
  • * High genetic polymorphism and mosaic expression in Ciona hinder efficient CRISPR/Cas9 knockout.
  • * In vitro approaches are needed to reduce animal use and overcome seasonality challenges in Ciona research.

Purpose of the Study:

  • * To optimize CRISPR/Cas9 gene editing protocols in Ciona intestinalis for improved efficiency and broader applicability.
  • * To develop in silico, in vitro, and in vivo strategies for more reliable CRISPR knockouts.
  • * To enhance the utility of Ciona as a model organism for genetic studies.

Main Methods:

  • * In silico: Correcting guide RNA (sgRNA) design by sequencing target regions and aligning with reference genomes to account for subspecies polymorphisms.
  • * In vitro: Validating sgRNA efficiency using in vitro assays to reduce in vivo experimentation.
  • * In vivo: Comparing Cas9 with Cas9:Geminin for enhanced editing efficiency, utilizing next-generation sequencing (NGS) for genotyping, and employing double CRISPR for large deletions.

Main Results:

  • * Optimized sgRNA design and in silico analysis improved accuracy by correcting for Ciona subspecies polymorphisms.
  • * In vitro assays showed suitability for sgRNA validation but also highlighted mismatch tolerance.
  • * Cas9:Geminin increased in vivo editing efficiency, enabling access to early expressed genes.
  • * Next-generation sequencing streamlined the identification of efficient sgRNAs, and double CRISPR generated significant deletions.

Conclusions:

  • * The presented improvements significantly enhance CRISPR/Cas9 efficiency and reliability in Ciona intestinalis.
  • * These optimized methods facilitate the establishment of CRISPR techniques in new research settings and are transferable to other organisms.
  • * The study provides a robust framework for advancing genetic engineering in Ciona and beyond.