Related Experiment Video
Updated: May 21, 2025
08:23
CIRCLE-Seq for Interrogation of Off-Target Gene Editing
Published on: November 1, 2024
452
Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing
Honglue Shi1, Noor Al-Sayyad2, Kevin M Wasko3
1Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
Molecular Cell
|April 24, 2025
Summary
CRISPR-Cas9 genome editing efficiency decreases with broader protospacer-adjacent motif (PAM) recognition. Reduced PAM specificity leads to non-selective DNA binding and lower editing success, highlighting a trade-off for improved editors.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- RNA-guided CRISPR-Cas enzymes are crucial for programmable genome editing.
- These enzymes recognize specific DNA sequences adjacent to a protospacer-adjacent motif (PAM).
Purpose of the Study:
- To investigate the molecular basis of high-efficiency genome editing.
- To understand how variations in protospacer-adjacent motif (PAM) binding specificity affect Streptococcus pyogenes Cas9 (SpyCas9) editing efficiency.
Main Methods:
- Biochemical assays
- Biophysical assays
- Cell-based assays
- Analysis of SpyCas9 variants with differing PAM specificities.
Main Results:
- Reduced PAM specificity resulted in persistent, non-selective DNA binding.
- This non-selective binding led to failures in stable guide RNA hybridization with the target DNA.
- Lowered genome-editing efficiency was observed in cells with reduced PAM specificity.
Conclusions:
- A trade-off exists between broad PAM recognition and genome-editing effectiveness.
- High-efficiency editing likely involves a two-step target capture: selective PAM binding followed by DNA unwinding.
- This model can guide the engineering of more effective CRISPR-Cas and RNA-guided genome editors.
Related Concept Videos
01:59CRISPR
48.8K
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...
48.8K
02:53Conservative Site-specific Recombination and Phase Variation
5.9K
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
5.9K
02:31Homologous Recombination
50.0K
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...
50.0K
02:53CRISPR and crRNAs
16.4K
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...
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...
16.4K