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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

186
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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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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Homologous Recombination02:31

Homologous Recombination

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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 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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Related Experiment Video

Updated: Sep 4, 2025

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
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Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

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Optimizing CRISPR/Cas9 Editing of Repetitive Single Nucleotide Variants.

Inga Usher1, Lorena Ligammari1, Sara Ahrabi2

  • 1Department of Pathology (Research), UCL Cancer Institute, University College London, London, United Kingdom.

Frontiers in Genome Editing
|July 22, 2022
PubMed
Summary

Optimizing CRISPR/Cas9 genome editing is crucial for research. This study found that while various factors influence editing efficiency, in silico predictions poorly correlate with actual cellular activity, highlighting the need for empirical validation.

Keywords:
CRISPRCRISPR/Cas9cell linegenome editinghomology directed repair (HDR)prime editingstem cells

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A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells
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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR/Cas9, base editors, and prime editors are key genome editing tools.
  • Optimizing CRISPR/Cas9 for homology directed repair (HDR)-mediated single nucleotide substitutions remains a significant challenge.
  • Existing studies often lack validation in clonal cell lines or for difficult genetic loci.

Purpose of the Study:

  • To investigate factors influencing CRISPR/Cas9 editing efficiency.
  • To compare the effectiveness of different optimization strategies.
  • To provide a validated protocol for improving genome editing outcomes.

Main Methods:

  • Conducted 95 transfections across two cell lines (colony forming and immortalized).
  • Evaluated effects of donor template modifications, component concentrations, HDR enhancers, and cold shock.
  • Utilized next-generation sequencing (NGS) and digital droplet PCR (ddPCR) for efficiency assessment.

Main Results:

  • In silico guide RNA efficiency predictions showed poor correlation with in vitro activity.
  • Observed editing efficiencies of 5-12% in transfected populations, decreasing to 1% upon clonal isolation.
  • Demonstrated significant variability in CRISPR efficiency based on cell model and target locus.

Conclusions:

  • Successful genome editing necessitates empirical comparison of systems and modifications for optimal protocols.
  • Validated HDR-boosting modifications for CRISPR/Cas9 are presented.
  • A flowchart is provided to guide researchers through the genome editing process.