Cas9-sgrna Rnp Complex
Cas9-sgrna Rnp Complex - Related Videos
02:15
Nanoblade-Based Delivery of Nucleic Acid Cargo: A Technique to Deliver Cas9-sgRNA Complex to Target Cells via Virus-Like Particles
0 Views •
2025
This video demonstrates a technique for nanoblade-based delivery of Cas9-sgRNA ribonucleoprotein complex inside target cells for genome editing. Nanoblades are virus-like particles devoid of the ability to multiply and infect neighboring cells, therefore are very useful for rapid and dose-dependent transportation of bio- and nanomaterials.
02:30
A CRISPR-Cas9 Technique for Gene Editing in T Cells
0 Views •
2025
This video demonstrates an assay for performing gene editing in human T cells using the CRISPR-Cas9 technology. A mixture of primary CD4+ and CD8+ T cells is combined with a CRISPR-Cas9 ribonucleoprotein complex, targeting specific genes for knockout. Upon electroporation, the sgRNA guides Cas9 to the target DNA sequence, creating precise cuts. These cuts are then repaired by the cell's non-homologous end-joining mechanism, leading to gene knockout.
06:12
Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex
0 Views •
Cited by 24 •
2018
Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.
09:23
Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles ("Nanoblades")
0 Views •
Cited by 6 •
2021
We have developed a simple and inexpensive protocol to load Cas9/single-guide RNA (sgRNA) ribonucleoprotein complexes within virus-like particles. These particles, called "Nanoblades", allow efficient delivery of the Cas9/sgRNA complex in immortalized and primary cells as well as in vivo.
04:51
High-Efficiency Gene Disruption in Primary Bone Marrow-Derived Macrophages Using Electroporated Cas9-sgRNA Complexes
0 Views •
2023
This protocol describes the procedure for genome editing in mouse bone marrow-derived macrophages using Cas9-sgRNA ribonucleoprotein complexes assembled in vitro and delivered by electroporation.
FAQs
The protospacer adjacent motif, or PAM, is a required DNA sequence feature that enables the Cas9-sgRNA RNP complex to act at a nearby target. Complementary pairing between the sgRNA and DNA identifies the intended sequence, but cleavage depends on the appropriate PAM context. Consequently, both sequence complementarity and PAM presence influence where genome editing can occur.
The sgRNA provides sequence recognition through complementary base pairing with the target DNA. This pairing positions the Cas9 nuclease near the selected region, where Cas9 can cleave both DNA strands close to the required PAM. In genetics experiments, changing the sgRNA sequence therefore redirects the complex toward different coding or regulatory regions.
After Cas9 cleaves both DNA strands, cellular DNA repair pathways process the resulting break and generate the sequence changes observed in the experiment. These repair-driven outcomes can support gene knockout or more precise genome editing, depending on the intended design and the genomic region being studied. The repair response therefore connects nuclease activity to the final genetic result.
A basic workflow uses a selected sgRNA together with Cas9, forms the RNP assembly, and delivers that preassembled complex to the genetic system under study. The complex then recognizes a complementary DNA sequence in a suitable PAM context and cleaves both strands. Subsequent cellular repair produces the targeted sequence changes that researchers analyze.
Researchers may choose this approach when they need to modify a defined coding or regulatory region for gene knockout, precise genome editing, or functional analysis. It is especially relevant when transient nuclease activity is desirable, because delivery as a preassembled RNP provides the active complex directly and can help limit prolonged exposure to Cas9.
By directing cleavage to selected coding or regulatory regions, the complex enables researchers to examine how specific genomic sequences contribute to gene function. Changes produced after cellular repair can be evaluated in functional studies, while coding-region disruption can support knockout experiments. Targeting regulatory regions extends the approach beyond altering protein-coding sequences.