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Related Concept Videos

Genetic Screens02:46

Genetic Screens

5.0K
Genetic screens are tools used to identify genes and mutations responsible for phenotypes of interest. Genetic screens help identify individuals or a group of people at risk of developing  genetic diseases and help them with early intervention, targeted therapy, and reproductive options.
Forward genetic screens
Forward or “classical” genetic screens involve creating random mutations in an organism’s DNA using radiation, mutagens, or insertion of additional bases, which...
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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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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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Related Experiment Video

Updated: Jul 31, 2025

Pooled CRISPR-Based Genetic Screens in Mammalian Cells
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Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Published on: September 4, 2019

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Optimized metrics for orthogonal combinatorial CRISPR screens.

Ronay Cetin1, Martin Wegner1, Leah Luwisch1

  • 1Institute of Biochemistry II, Faculty of Medicine, Goethe University Frankfurt, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany.

Scientific Reports
|May 6, 2023
PubMed
Summary
This summary is machine-generated.

Choosing the right CRISPR-associated (Cas) nuclease is crucial for large-scale gene dependency mapping. This study found CRISPR-SpCas9 superior to AsCas12a for combinatorial screens, and developed a novel multiplex approach for orthogonal gene editing.

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Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
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Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR-based gene perturbation is vital for studying genotype-phenotype links.
  • Efficient CRISPR nucleases are essential for large-scale combinatorial and orthogonal screening.
  • Systematic comparisons of commonly used CRISPR nucleases like SpCas9 and AsCas12a are limited.

Purpose of the Study:

  • To systematically compare the performance of SpCas9, AsCas12a, and CHyMErA for combinatorial and orthogonal CRISPR screens.
  • To identify critical performance parameters for selecting CRISPR nucleases.
  • To engineer improved CRISPR systems for orthogonal applications.

Main Methods:

  • Comparative analysis of SpCas9, AsCas12a, and CHyMErA in hTERT-immortalized retinal pigment epithelial cells.
  • Systematic evaluation of combinatorial and orthogonal screening capabilities.
  • Engineering of dual-guide RNAs (gRNAs) and a novel multiplex SpCas9-enAsCas12a (multiSPAS) system.

Main Results:

  • SpCas9 demonstrated superior performance over enhanced and optimized AsCas12a in combinatorial screens.
  • CHyMErA showed limited activity under tested conditions.
  • Dual-gRNAs improved CHyMErA performance but negatively impacted AsCas12a's effect size range.
  • The multiSPAS system enabled efficient orthogonal gene editing without RNA processing.

Conclusions:

  • SpCas9 is a more robust choice than AsCas12a for combinatorial CRISPR screens.
  • The multiSPAS system offers an effective solution for orthogonal CRISPR applications.
  • Optimized CRISPR-nuclease selection and system engineering are key for advancing large-scale genetic screens.