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Related Concept Videos

CRISPR01:59

CRISPR

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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 and crRNAs02:53

CRISPR and crRNAs

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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.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
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The Antiviral System of Bacteria and Archaea: CRISPR01:23

The Antiviral System of Bacteria and Archaea: CRISPR

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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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Genetic Screens02:46

Genetic Screens

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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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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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Cancers Originate from Somatic Mutations in a Single Cell02:21

Cancers Originate from Somatic Mutations in a Single Cell

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Cancer arises from mutations in the critical genes that allow healthy cells to escape cell cycle regulation and acquire the ability to proliferate indefinitely. Though originating from a single mutation event in one of the originator cells, cancer progresses when the mutant cell lines continue to gain more and more mutations, and finally, become malignant. For example, chronic myelogenous leukemia (CML) develops initially as a non-lethal increase in white blood cells, which progressively...
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Pooled CRISPR-Based Genetic Screens in Mammalian Cells
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CRISPR Screening in Single Cells.

Johan Henriksson1

  • 1Molecular Infection Medicine Sweden, Umeå University, Umeå, Sweden. mahogny@areta.org.

Methods in Molecular Biology (Clifton, N.J.)
|April 28, 2019
PubMed
Summary
This summary is machine-generated.

Single-cell RNA sequencing combined with CRISPR screening enables comprehensive gene analysis. This study details protocols for CRISPR screening in single cells, covering cloning, virus production, and sequencing data generation.

Keywords:
10×CRISPRCRISPRiCloningDropletsLentivirusMultiplexingPoolingRNA-seqSingle-cellTransductionTransfectionVirus production

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Area of Science:

  • Genomics
  • Molecular Biology
  • Biotechnology

Background:

  • CRISPR screening is a powerful tool for functional genomics.
  • Single-cell RNA sequencing (scRNA-seq) provides high-resolution gene expression data.
  • Integrating these technologies offers unprecedented insights into gene function.

Purpose of the Study:

  • To describe current protocols for CRISPR screening in single cells.
  • To outline the workflow from gene targeting to sequencing data generation.
  • To facilitate the application of CRISPR screening in single-cell studies.

Main Methods:

  • CRISPR library design and cloning.
  • Lentivirus production for gene delivery.
  • Single-cell isolation and barcoding.
  • RNA extraction, library preparation, and sequencing.
  • Bioinformatic analysis of sequencing data.

Main Results:

  • Established robust protocols for CRISPR screening in single cells.
  • Demonstrated feasibility across various cell types and experimental scales.
  • Generated high-quality sequencing data suitable for downstream analysis.

Conclusions:

  • The described protocols enable efficient, large-scale interrogation of gene function at the single-cell level.
  • This integrated approach expands the scope of genetic screening.
  • Facilitates discovery of gene roles in cellular processes and disease.