Cas9-mediated Cleavage
Cas9-mediated Cleavage - Related Videos
03:34
Assessing Caspase-Mediated Cleavage of Viral and Host Proteins in Virus-Infected Cells
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2025
The video demonstrates a method to assess caspase-mediated protein cleavage in virus-infected cells. Post-viral infection, caspases get activated and degrade various host cellular proteins and viral nucleoproteins while specific caspase inhibitors prevent this degradation, as confirmed by western blotting.
11:35
Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
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Cited by 6 •
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Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.
01:33
Cleavage and Blastulation
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2019
After a large-single-celled zygote is produced via fertilization, the process of cleavage occurs while zygotes travel through the uterine tube. Cleavage is a mitotic cell division that does not result in growth. With each round of successive cell division, daughter cells get increasingly smaller. Zygotic Genome Activation At the beginning of embryogenesis, maternal mRNAs control development. However, by the eight-cell stage of cleavage, embryonic genes become activated in a process called...
07:33
Physical Properties Of Minerals I: Crystals and Cleavage
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2023
Source: Laboratory of Alan Lester - University of Colorado Boulder The physical properties of minerals comprise various measurable and discernible attributes, including color, streak, magnetic properties, hardness, crystal growth form, and crystal cleavage. Each of these properties are mineral-specific, and they are fundamentally related to a particular mineral’s chemical make-up and atomic structure. This experiment examines two properties that stem primarily from symmetric repetition of...
10:37
CRISPR/Cas9-mediated Endogenous Fluorescent Tagging of Germline-specific Genes in Caenorhabditis elegans
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2026
A CRISPR/Cas9 microinjection workflow for endogenous fluorescent tagging in the Caenorhabditis elegans germline to obtain homozygous knock-in lines for in vivo protein localization analysis.
FAQs
The protospacer-adjacent motif, or PAM, provides a compatibility check next to the intended DNA target. Guide-RNA pairing alone is not sufficient in the described reaction because Cas9 recognizes the complementary sequence in the context of a compatible PAM. Consequently, PAM presence helps determine whether a selected site can be engaged and cleaved by the Cas9-guide RNA complex.
Guide-RNA pairing directs Cas9 toward a complementary genetic sequence, making sequence recognition the central determinant of targeting. If related sequences are also recognized, cleavage specificity can decrease and off-target activity may occur. Biochemical studies therefore examine guide-RNA recognition alongside sequence complementarity to understand why some targets are selected more precisely than others.
After guide RNA pairing identifies a compatible target, Cas9 activates its nuclease domains. These catalytic components sever both DNA strands rather than cutting only one strand, producing a site-specific double-strand break. The break is the critical biochemical intermediate that connects target recognition with subsequent genome-editing outcomes mediated by cellular DNA-repair pathways.
The overview identifies guide-RNA recognition, nuclease specificity, and reaction conditions that influence editing efficiency and off-target activity. Target sequence pairing and the presence of a compatible PAM affect whether cleavage is directed to a selected site, while Cas9 nuclease behavior affects how selectively the DNA is cut. These variables are central to evaluating cleavage performance.
A basic workflow begins by selecting a genetic sequence that can pair with a guide RNA and lies next to a compatible PAM. The guide RNA then directs Cas9 to that site, where the nuclease domains generate a double-strand break. Researchers next evaluate how cellular DNA-repair pathways process the break and what editing outcome results.
Cas9 cutting creates a double-strand break, but the eventual genetic result depends on how the cell repairs that lesion. In the described applications, repair can support targeted gene disruption or sequence insertion. Thus, cleavage establishes the programmable DNA lesion, whereas cellular repair pathways help determine the specific genome-editing outcome observed after treatment.
The reaction provides a system for studying how a protein nuclease, a guide RNA, and a DNA target work together at the molecular level. Biochemistry researchers can use it to investigate guide-RNA recognition, nuclease specificity, cleavage efficiency, and off-target activity. The same principles support targeted gene disruption, sequence insertion, and broader genome-editing studies.