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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 interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.
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CIRCLE-Seq for Interrogation of Off-Target Gene Editing
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Seed sequences mediate off-target activity in the CRISPR-interference system.

Neha Rohatgi1, Jean-Philippe Fortin2, Ted Lau3

  • 1Genentech Computational Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA; Roche Informatics, Hoffman-La Roche Canada, 7070 Mississauga Road, Mississauga, ON, Canada.

Cell Genomics
|November 7, 2024
PubMed
Summary
This summary is machine-generated.

The CRISPR interference (CRISPRi) system can cause widespread gene silencing off-target effects. These unintended impacts are mainly due to seed sequence complementarity between the sgRNA and genomic DNA.

Keywords:
CRISPR activationCRISPR interferencePAM-proximal seed sequenceoff-target activity

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Area of Science:

  • Molecular Biology
  • Genetics
  • Functional Genomics

Background:

  • CRISPR interference (CRISPRi) is a key technology for gene silencing in research.
  • Systematic investigation of CRISPRi off-target activity is lacking.

Purpose of the Study:

  • To investigate genome-wide off-target activity of the CRISPRi system.
  • To understand the impact of off-target effects on gene expression.

Main Methods:

  • Utilized a genome-wide CRISPRi-Cas9 single-guide RNA (sgRNA) library.
  • Analyzed off-target activity and its effects on gene expression.

Main Results:

  • Off-target effects in CRISPRi are pervasive, impacting gene expression directly and indirectly.
  • Most off-targets are linked to the seed sequence (3' half of sgRNA spacer) complementarity with PAM-proximal genomic regions.
  • Off-target binding stability is influenced by seed sequence length and mismatch tolerance.

Conclusions:

  • CRISPRi off-target activity is a significant consideration in functional genomics.
  • Understanding seed sequence interactions is crucial for predicting and mitigating CRISPRi off-target effects.