Related Experiment Video
Updated: Jun 17, 2025
11:53
Substrate Generation for Endonucleases of CRISPR/Cas Systems
Published on: September 8, 2012
27.3K
Structural determinants of DNA cleavage by a CRISPR HNH-Cascade system
Seiichi Hirano1, Han Altae-Tran1, Soumya Kannan1
1Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Cambridge, MA 02139, USA.
Molecular Cell
|August 7, 2024
Summary
A novel CRISPR-Cas system (HNH-Cascade) precisely cleaves DNA without Cas3, using an integrated HNH endonuclease. This discovery reveals a unique hybrid molecular machine for programmable DNA targeting and cleavage.
Area of Science:
- Molecular Biology
- Microbial Genetics
- Structural Biology
Background:
- Prokaryotic type I CRISPR-Cas systems use Cascade complexes and Cas3 for DNA degradation.
- A subtype I-F1 CRISPR-Cas system (HNH-Cascade) lacks Cas3 but possesses an HNH endonuclease domain within its Cas8 component.
Purpose of the Study:
- To determine the cryo-EM structure of Selenomonas sp. HNH-Cascade (SsCascade) bound to target DNA.
- To characterize the mechanism of action of this Cas3-deficient CRISPR-Cas system.
Main Methods:
- Cryo-electron microscopy (cryo-EM) to resolve the SsCascade-DNA complex structure.
- Biochemical assays to characterize the nuclease activity and mechanism.
Main Results:
- The cryo-EM structure reveals a ring-like SsCascade complex.
- The integrated HNH domain precisely cleaves unwound target DNA.
- SsCascade functions as a hybrid of Cascade and HNH nuclease.
Conclusions:
- HNH-Cascade represents a distinct CRISPR-Cas subtype with a unique DNA cleavage mechanism.
- This system offers a Cas3-independent platform for programmable DNA manipulation.
- The structural and mechanistic insights advance our understanding of CRISPR-Cas evolution and function.
Related Concept Videos
02:53CRISPR and crRNAs
16.9K
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...
16.9K
01:59CRISPR
50.1K
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...
50.1K
02:31Homologous Recombination
50.4K
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...
50.4K
01:11Restriction Enzymes
29.6K
Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called restriction sites. Such palindromic DNA sequences or inverted repeats are commonly found in regions of functional significance, such as the origin of replication, gene operator sites, and regions containing transcription termination signals.
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...
29.6K
01:03Single-Strand DNA Binding Proteins
14.0K
For successful DNA replication, the unwinding of double-stranded DNA must be accompanied by stabilization and protection of the separated single strands of the DNA. This crucial task is performed by single-strand DNA-binding (SSB) proteins. They bind to the DNA in a sequence-independent manner, which means that the nitrogenous bases of the DNA need not be present in a specific order for binding of SSB proteins to it. The binding of SSB proteins straightens single-stranded DNA (ssDNA) and makes...
14.0K
02:04Fixing Double-strand Breaks
12.5K
The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the back-up in case the other strand is damaged. Second, the double-helical structure can be wrapped around proteins called histones to form nucleosomes, which can then be tightly wound to form chromosomes. This way, DNA chains up to 2 inches long can be contained within microscopic structures in a cell. A double-stranded break not only damages...
12.5K