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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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CRISPR01:59

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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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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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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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Genome Editing in Mammalian Cell Lines using CRISPR-Cas
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SNP-CRISPR: A Web Tool for SNP-Specific Genome Editing.

Chiao-Lin Chen1, Jonathan Rodiger1,2, Verena Chung1,2

  • 1Department of Genetics.

G3 (Bethesda, Md.)
|December 12, 2019
PubMed
Summary

SNP-CRISPR is a new web tool for designing single guide RNAs (sgRNAs) for CRISPR-Cas9 genome editing. It effectively targets genetic mutations and single nucleotide polymorphisms (SNPs) in various genomes and genetic backgrounds.

Keywords:
CRISPRgenome editinggenome variant

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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:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • CRISPR-Cas9 technology relies on single guide RNA (sgRNA) for target specificity.
  • Existing sgRNA design tools are limited by reference genomes and do not account for genetic variations like SNPs.
  • SNPs can disrupt guide-target complementarity, reducing CRISPR editing efficiency.

Purpose of the Study:

  • To develop a web tool, SNP-CRISPR, for designing sgRNAs that accommodate genetic variations.
  • To enable sgRNA design for non-reference genomes, diverse genetic backgrounds, and specific SNP targeting.
  • To facilitate the correction of disease-associated mutations using CRISPR-Cas9.

Main Methods:

  • Developed the SNP-CRISPR web tool (https://www.flyrnai.org/tools/snp_crispr/).
  • Integrated public variant datasets and user-identified variants for sgRNA design.
  • Implemented algorithms to compute efficiency and specificity scores for sgRNAs targeting variants and reference sequences.
  • Enabled simultaneous targeting of multiple nearby base changes with a single sgRNA design.

Main Results:

  • SNP-CRISPR facilitates identification of effective sgRNAs in non-reference genomes and across varying genetic backgrounds.
  • The tool accurately designs sgRNAs for targeting specific SNP-containing alleles, including disease-relevant mutations.
  • SNP-CRISPR provides efficiency and specificity scores, aiding in optimal sgRNA selection.
  • The tool supports the design of sgRNAs to target multiple nearby base changes concurrently.

Conclusions:

  • SNP-CRISPR enhances the utility of CRISPR-Cas9 technology by addressing limitations posed by genetic variations.
  • The tool offers a versatile solution for designing sgRNAs in diverse research applications, including model systems.
  • SNP-CRISPR is valuable for designing sgRNAs for precise correction of disease-associated variants.