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CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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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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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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CRISPR/Cas9 Targeted Mutagenesis for Functional Genetics in Maize.

Charles T Hunter1

  • 1Chemistry Research Unit, USDA Agricultural Research Service, Gainesville, FL 32608, USA.

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|April 30, 2021
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Summary

This study details using CRISPR/Cas9 for precise gene editing in maize, enabling efficient creation of loss-of-function mutations. It introduces a new vector for simultaneous multiple guide RNA delivery, streamlining mutant isolation.

Keywords:
gene editinggene knockoutpolycistronic gRNAtransformation vector

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Area of Science:

  • Plant genetics
  • Molecular biology
  • Biotechnology

Background:

  • CRISPR/Cas9 is a key tool for plant functional genetics.
  • It enables precise generation of loss-of-function alleles.
  • Efficient methods are needed for maize gene editing.

Purpose of the Study:

  • To provide guidelines for CRISPR/Cas9 experimental design in maize.
  • To introduce a modified vector for multiplexed gRNA delivery.
  • To optimize the isolation of homozygous mutants.

Main Methods:

  • Utilizing the CRISPR/Cas9 system for targeted mutagenesis in maize.
  • Employing a modified pRGEB32-BAR vector for simultaneous multiple guide RNA delivery.
  • Implementing efficient genetic strategies and genotyping approaches.

Main Results:

  • Demonstrated successful application of CRISPR/Cas9 for maize mutagenesis.
  • Validated a novel vector system for multiplexed gRNA delivery.
  • Established time- and cost-efficient methods for isolating homozygous mutants.

Conclusions:

  • CRISPR/Cas9 is a powerful and accessible tool for maize functional genomics.
  • The described vector and strategies facilitate efficient mutant generation.
  • This work aids researchers in accelerating maize genetic studies.