Homing Endonucleases
Homing Endonucleases - Related Videos
10:40
Homing of Hematopoietic Cells to the Bone Marrow
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Cited by 15 •
2009
This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.
11:53
Substrate Generation for Endonucleases of CRISPR/Cas Systems
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Cited by 4 •
2012
CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.
07:42
Non-invasive Imaging of Leukocyte Homing and Migration in vivo
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Cited by 8 •
2010
Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.
10:25
Competitive Homing Assays to Study Gut-tropic T Cell Migration
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Cited by 11 •
2011
Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.
09:40
Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae
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Cited by 9 •
2011
The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation.
FAQs
The process couples cleavage with repair. After the enzyme cuts the matching site on one chromosome, homologous recombination can use the corresponding intact chromosome as a template. During that repair event, the endonuclease gene is copied into the chromosome that was initially cut, converting an unmodified site into one carrying the enzyme gene.
Recognition of long DNA sequences contributes to the enzymes’ high sequence specificity. This specificity helps distinguish the intended genomic site from unrelated sequences, which is valuable for targeted modification. However, a suitable matching sequence must exist in the genome, so target-site availability remains a practical constraint when planning an engineering strategy.
Homologous recombination supplies the repair route that enables gene insertion after cleavage. The intact homologous DNA serves as the template for repairing the double-strand break, allowing sequence information that includes the endonuclease gene to be copied into the cut chromosome. Consequently, cleavage alone is insufficient for the copying outcome.
Their natural combination of extended sequence recognition, targeted double-strand cleavage, and repair-associated gene copying provides a model for engineering related genome-modification systems. This biological behavior informs nuclease design and gene-drive research, while also highlighting the need to manage target-site availability and maintain accurate control over the repair process.
A general workflow begins by identifying a genomic site that matches the enzyme’s recognition requirements. The enzyme then creates a double-strand break at that location, after which an intact homologous sequence can guide repair. The resulting chromosome is evaluated for copying of the endonuclease gene and the intended genome-integration outcome.
Success depends on more than cleavage. The genome must contain an appropriate matching target, and repair must proceed through homologous recombination with an intact homologous template. Accurate control of that repair is therefore essential. If target availability or repair control is inadequate, the intended gene-copying and genome-modification outcomes may not occur.
These enzymes support several bioengineering applications, including gene targeting, genome integration, synthetic biology, and investigations of DNA repair. They are particularly informative when researchers need to connect a defined DNA break with homologous-recombination-based sequence insertion. Their behavior also provides scientific context for developing engineered nucleases and studying gene-drive systems.