Related Experiment Video
Updated: Aug 2, 2025
11:53
Substrate Generation for Endonucleases of CRISPR/Cas Systems
Published on: September 8, 2012
27.4K
Novel molecular requirements for CRISPR RNA-guided transposition
Matt W G Walker1, Sanne E Klompe2, Dennis J Zhang1
1Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Nucleic Acids Research
|April 20, 2023
Summary
CRISPR-associated transposases (CASTs) facilitate DNA integration. This study identifies key DNA sequence requirements and reveals that integration host factor (IHF) is essential for efficient VchCAST transposition.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR-associated transposases (CASTs) are RNA-guided systems for DNA integration.
- Understanding sequence determinants is crucial for optimizing CAST-mediated transposition.
Purpose of the Study:
- To identify sequence requirements for efficient DNA integration by the Vibrio cholerae CAST system (VchCAST).
- To explore the role of cellular factors in VchCAST transposition.
- To engineer VchCAST for genome engineering applications.
Main Methods:
- Pooled library screening of transposon ends and target sites.
- High-throughput sequencing to analyze sequence preferences.
- Design and testing of modified transposon variants.
Main Results:
- Identified nucleotide preferences for the TnsB transposase on donor DNA.
- Discovered a requirement for integration host factor (IHF) in VchCAST transposition.
- Uncovered specific sequence motifs preferred at target integration sites.
- Developed in-frame protein tagging transposon variants.
Conclusions:
- Novel sequence determinants for VchCAST transposition were revealed.
- Integration host factor (IHF) is a novel cellular factor essential for VchCAST function.
- Findings inform the design of CAST systems for genome engineering.
Related Concept Videos
02:13Overview of Transposition and Recombination
15.9K
Transposons make up a significant part of genomes of various organisms. Therefore, it is believed that transposition played a major evolutionary role in speciation by changing genome sizes and modifying gene expression patterns. For example, in bacteria, transposition can lead to conferring antibiotic resistance. Movement of transposable elements within the genetic pool of pathogenic bacteria can aid in transfer of antibiotic-resistant genetic elements. In eukaryotes, transposons can carry out...
15.9K
02:57DNA-only Transposons
14.6K
DNA-only transposons are called autonomous transposons since they code for the enzyme transposase that is required for the transposition mechanism. Insertion of transposons can alter gene functions in multiple ways. They can mutate the gene, alter gene expression by introducing a novel promoter or insulator sequence, introduce new splice sites, and change the mRNA transcripts produced, or remodel chromatin structure.
The donor site from where the transposon is excised is either degraded or...
The donor site from where the transposon is excised is either degraded or...
14.6K
01:59CRISPR
52.5K
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...
52.5K
02:53CRISPR and crRNAs
17.1K
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...
17.1K
02:57piRNA - Piwi-interacting RNAs
6.9K
PIWI-interacting RNAs, or piRNAs, are the most abundant short non-coding RNAs. More than 20,000 genes have been found in humans that code for piRNAs while only 2000 genes have been found for miRNAs. piRNAs can act at the transcriptional and post-transcriptional levels and have a vital role in silencing transposable elements present in germ cells. They are also involved in epigenetic silencing and activation. Previously, they were thought to function only in germ cells but new evidence suggests...
6.9K
02:53Conservative Site-specific Recombination and Phase Variation
6.0K
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...
6.0K