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Related Concept Videos

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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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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 stands for Clustered Regularly Interspaced Short Palindromic Repeats is a adaptive immune system found in bacteria and archaea that protects against viral infections. This system enables prokaryotic cells to identify, remember, and neutralize foreign genetic elements, primarily bacteriophages, by storing fragments of the invader’s DNA as a genetic memory.The CRISPR immune response begins during an initial infection. Cas (CRISPR-associated) proteins play a central role in this...
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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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CRISPR-SE: a brute force search engine for CRISPR design.

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CRISPR-SE accurately identifies all off-target sites for CRISPR genome editing, unlike standard alignment tools that miss ultra-short sequences. This improves the reliability of CRISPR-Cas9 designs.

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Area of Science:

  • Genomics
  • Biotechnology
  • Bioinformatics

Background:

  • CRISPR genome editing relies on accurate identification of off-target sites.
  • Existing CRISPR design tools often use sequence alignment tools (e.g., BLAST, BWA) to find off-targets.
  • These alignment tools may fail to detect off-targets for short sequences like 20-bp guide RNAs (gRNAs) due to their algorithms.

Purpose of the Study:

  • To evaluate the accuracy of existing search engines for CRISPR off-target identification.
  • To introduce CRISPR-SE, a novel, accurate, and fast search engine for CRISPR off-target analysis.

Main Methods:

  • Developed four sets of gRNAs to test existing search engines.
  • Implemented CRISPR-SE, a brute-force search engine that virtually compares all gRNAs against a query gRNA.
  • Performed accuracy benchmarking comparing CRISPR-SE with multiple alignment-based search engines.

Main Results:

  • Alignment tools provided incomplete and inconsistent lists of off-target sites, especially for ultra-short sequences.
  • CRISPR-SE demonstrated high accuracy in identifying all potential off-target sites.
  • CRISPR-SE achieved competitive speed performance.

Conclusions:

  • Standard alignment tools are inadequate for comprehensive CRISPR off-target prediction due to algorithmic limitations.
  • CRISPR-SE offers a robust solution for accurate and efficient off-target identification.
  • CRISPR-SE is expected to enhance the quality and reliability of CRISPR-based genome engineering designs.