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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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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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A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization
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A Novel CRISPR Interference Effector Enabling Functional Gene Characterization with Synthetic Guide RNAs.

Clarence Mills1, Andrew Riching1, Ashleigh Keller1

  • 1Horizon Discovery, a PerkinElmer Company, Lafayette, Colorado, USA and University of Colorado-Boulder, Boulder, Colorado, USA.

The CRISPR Journal
|October 18, 2022
PubMed
Summary
This summary is machine-generated.

A new CRISPR interference (CRISPRi) system, dCas9-SALL1-SDS3, enhances gene repression using synthetic guide RNAs. This advancement enables precise gene characterization in arrayed screening formats, including primary cells.

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Area of Science:

  • Molecular Biology
  • Gene Regulation
  • CRISPR Technology

Background:

  • CRISPR interference (CRISPRi) is established for pooled lentiviral screening.
  • Limited application of CRISPRi with synthetic guide RNAs in arrayed screening formats.
  • Need for enhanced gene repression and specificity in CRISPRi systems.

Purpose of the Study:

  • To develop a novel CRISPRi system for arrayed screening.
  • To improve target gene repression and specificity using synthetic guide RNAs.
  • To enable functional gene characterization in primary cells.

Main Methods:

  • Development of a deactivated Cas9 fusion protein (dCas9-SALL1-SDS3).
  • Utilizing chemically modified synthetic single guide RNAs (sgRNAs).
  • In vitro transcription of dCas9-SALL1-SDS3 mRNA for transient delivery.
  • Application in arrayed-format screening for DNA damage host factors.

Main Results:

  • dCas9-SALL1-SDS3 demonstrated superior gene repression compared to previous CRISPRi systems.
  • High target specificity was observed with synthetic sgRNAs.
  • Successful interaction of dCas9-SALL1-SDS3 with histone deacetylase and Swi-independent three complexes.
  • Effective delivery and application in primary cells, including human induced pluripotent stem cells and T cells.

Conclusions:

  • dCas9-SALL1-SDS3 represents a significant advancement in CRISPRi technology for arrayed screening.
  • The system offers enhanced gene repression and specificity, suitable for complex phenotypic readouts.
  • Enables short-term gene modulation in primary cells, expanding CRISPRi applications.