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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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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.
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RNA editing is a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the...
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Genomics is the science of genomes: it is the study of all the genetic material of an organism. In humans, the genome consists of information carried in 23 pairs of chromosomes in the nucleus, as well as mitochondrial DNA. In genomics, both coding and non-coding DNA is sequenced and analyzed. Genomics allows a better understanding of all living things, their evolution, and their diversity. It has a myriad of uses: for example, to build phylogenetic trees, to improve productivity and...
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Diploid organisms inherit genetic material through chromosomes from both parents. Copies of the same gene are known as alleles. In most cases, both alleles are simultaneously expressed and allow various cellular processes to function optimally. If one of the alleles is missing or mutated, the expression of the other allele can compensate; however, this is not true for all genes.
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Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish
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Genome Editing: CRISPR-Cas9.

Jakob B Hoof1, Christina S Nødvig1, Uffe H Mortensen2

  • 1Department of Biotechnology and Biomedicine, Technical University of Denmark, Kgs.Lyngby, Denmark.

Methods in Molecular Biology (Clifton, N.J.)
|June 8, 2018
PubMed
Summary
This summary is machine-generated.

This chapter provides protocols for CRISPR-Cas9 gene editing in fungi lacking genetic tools. It details marker identification, vector construction, reporter gene mutagenesis, and gene targeting strategies for efficient genetic engineering.

Keywords:
CRISPRGene editingGene targetingMutagenesisNonmodel fungi

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Area of Science:

  • Molecular Biology
  • Mycology
  • Genetic Engineering

Background:

  • CRISPR-Cas9 technology offers powerful gene editing capabilities.
  • Fungi often lack established genetic tools, hindering research and application.
  • Efficient genetic engineering in fungi is crucial for various biological studies.

Purpose of the Study:

  • To provide comprehensive protocols for implementing CRISPR-Cas9 in fungi with limited genetic tools.
  • To establish guidelines for successful CRISPR-Cas9 mediated gene targeting and engineering in fungal species.

Main Methods:

  • Identification of dominant markers for genetic transformation.
  • Construction of Cas9/sgRNA episomal expression vectors.
  • Mutagenesis of reporter genes to assess CRISPR-Cas9 efficiency.
  • Development of CRISPR-mediated marker-dependent and marker-free gene targeting.

Main Results:

  • Established protocols for CRISPR-Cas9 implementation in under-resourced fungal systems.
  • Demonstrated methods for vector construction and gene targeting.
  • Provided a framework for assessing and optimizing CRISPR-Cas9 efficiency in fungi.

Conclusions:

  • This chapter facilitates the adoption of CRISPR-Cas9 technology in diverse fungal species.
  • The presented guidelines enable efficient genetic engineering and gene targeting in fungi.
  • Empowers researchers to utilize advanced gene editing tools in mycology.