Primary Cell Editing
Primary Cell Editing - Related Videos
09:53
Gene Editing of Primary Rhesus Macaque B Cells
0 Views •
Cited by 12 •
2023
We present a method for culturing and gene editing primary rhesus macaque B cells using CRISPR/Cas9 and recombinant adeno-associated virus serotype 6 for the study of B cell therapies.
10:36
Genome Editing in Primary Mammalian Cells via Electroporation of Editor RNA
0 Views •
2026
The delivery of genome editors as RNAs is a particularly potent means of modifying the genomes of primary mammalian cells and cell lines. The goal of this protocol is to deliver genome editors as mRNA into primary mammalian cells and cell lines, followed by targeted Illumina sequencing and analysis to quantify editing outcomes.
09:16
Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System
0 Views •
Cited by 4 •
2017
This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.
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.
08:31
Genome Editing
0 Views •
2023
A well-established technique for modifying specific sequences in the genome is gene targeting by homologous recombination, but this method can be laborious and only works in certain organisms. Recent advances have led to the development of “genome editing”, which works by inducing double-strand breaks in DNA using engineered nuclease enzymes guided to target genomic sites by either proteins or RNAs that recognize specific sequences. When a cell attempts to repair this damage, mutations can be...
FAQs
In a CRISPR-based workflow, the guide RNA identifies a selected DNA sequence and directs the nuclease to that location. The nuclease then creates a targeted cleavage, initiating cellular repair. This two-part targeting mechanism connects sequence selection with the cell’s own repair response, allowing researchers to investigate the function of specific genes in donor-derived cellular systems.
Nonhomologous end joining and homology-directed repair are cellular pathways that act after nuclease-mediated DNA cleavage. Because repair follows targeting, editing results depend on both the selected DNA site and the repair conditions available in the cells. Optimizing repair conditions is particularly important when primary cells are difficult to maintain, transfect, or expand.
Delivery efficiency, cell viability, and repair conditions are central variables in primary cell editing. A delivery approach must introduce the editing components while preserving enough viable cells for the experiment. At the same time, the cells must support the relevant repair response. These constraints make optimization necessary before interpreting gene-function or disease-modeling results.
Primary cells isolated directly from tissues retain many donor-specific properties, which can make experimental findings more representative of physiologically relevant cellular systems. Editing these cells therefore supports investigations in a context that may preserve characteristics lost during extensive cell expansion. This feature is especially valuable when researchers need to connect a genetic change with cellular behavior linked to a particular donor.
A typical workflow begins with cells isolated directly from tissue, followed by delivery of a guide RNA and nuclease system. The guide directs cleavage at the selected DNA sequence, after which the cells repair the break through nonhomologous end joining or homology-directed repair. Researchers must optimize delivery, viability, and repair conditions throughout the workflow because primary cells can be difficult to maintain and expand.
Researchers use primary cell editing when they need to study gene function in cells that retain donor-specific properties. The approach supports disease modeling, functional genomics, cell therapy research, and validation of genetic variants. These applications benefit from examining genetic changes in cellular systems that remain closer to the physiological context of the tissue from which the cells were obtained.
In genetics research, edited primary cells can provide a setting for testing how selected genetic changes affect cellular systems. The method is useful for validating genetic variants, modeling disease-related biology, and examining gene function through targeted DNA modification. Because the cells originate directly from tissue and retain donor-specific properties, results can be interpreted in a physiologically relevant context.