Single Guide Rna
Single Guide Rna - Related Videos
06:59
Nanomanipulation of Single RNA Molecules by Optical Tweezers
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Cited by 13 •
2014
Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5’ and 3’ ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.
03:58
Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System
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Cited by 6 •
2023
In this protocol, we describe the technical procedures to generate brown adipose tissue (BAT)-specific knockout mice leveraging a combined Cre-LoxP, CRISPR-Cas9, and adeno-associated virus (AAV) single-guide RNA (sgRNA) system. The described steps include the design of the sgRNAs, the preparation of the AAV-sgRNA particles, and the microinjection of AAV into the BAT lobes.
06:48
CRISPR Guide RNA Cloning for Mammalian Systems
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Cited by 16 •
2018
Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.
14:53
Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia
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Cited by 2 •
2014
We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.
09:56
A Stepwise Guide to the Isolation and Analysis of Leaf Surface and Apoplastic RNA Using Arabidopsis Rosettes
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2025
We describe a simple yet highly reproducible method to isolate extracellular RNA from the leaf surface and apoplast of Arabidopsis plants.
FAQs
The guide sequence provides recognition by pairing with a complementary DNA target, while the scaffold forms the structural region that binds the CRISPR-associated protein Cas9. Both parts are required for coordinated targeting: the guide supplies sequence selectivity, and the scaffold supports protein association. This division allows researchers to alter target recognition while retaining Cas9-compatible structural features.
A protospacer adjacent motif, or PAM, is a nearby DNA sequence required for Cas9-compatible targeting. Guide complementarity alone is therefore insufficient if the selected region lacks an appropriate PAM. During design, researchers must consider both the intended complementary sequence and the neighboring PAM, because this combination determines whether Cas9 can act at the chosen genomic site.
Complementarity connects the guide portion of the RNA with the selected nucleic acid sequence, helping determine where the CRISPR-Cas9 system acts. When targeting conditions are met, Cas9 produces a site-specific double-strand break at that location. The selected target therefore influences which gene or regulatory region is modified and what biological question the experiment can address.
Design begins by selecting a nucleic acid region relevant to the research question, identifying a compatible PAM, and specifying a guide sequence complementary to the target. The guide must also be incorporated with the scaffold region that binds Cas9. These choices connect the intended genomic site with the desired experimental purpose, such as gene disruption or sequence insertion.
Depending on the CRISPR system and experimental design, targeting can support gene knockout, targeted sequence insertion, transcriptional regulation, or genome screening. A knockout investigates consequences of disrupting a gene, whereas insertion adds a selected sequence. Regulation changes gene expression without necessarily using the same outcome as a sequence modification, allowing distinct functional questions to be tested.
Their programmable targeting lets researchers examine the effects of altering selected genes or genomic regions in cells and organisms. Experiments can connect a targeted genetic change with gene function, disease mechanisms, or broader biological responses. Genome screening extends this approach across many targets, helping identify genes or sequences associated with a phenotype or process under study.