Gene Knockin
Gene Knockin - Related Videos
08:25
Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli
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Cited by 5 •
2016
We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.
11:32
Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines
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Cited by 7 •
2021
This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.
08:48
Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice
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Cited by 13 •
2022
The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.
05:12
All-in-One CRISPR Genome Editing: A Method for Homology Directed Repair-Based Gene Knock-In in Cultured Cells Using CRISPR-Cas9 System
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2025
In this video, we demonstrate all-in-one CRISPR-Cas9 based genome editing in cultured cells where Cas9 and sgRNA are provided as a single plasmid construct to the cells. The CRISPR-Cas9 system and desired gene to be inserted was introduced in cells through electroporation technique to facilitate successful gene editing.
10:12
Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing
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Cited by 5 •
2022
The protocol describes how to generate knock-out myoblasts using CRISPR/Cas9, starting from the design of guide-RNAs to the cellular cloning and characterization of the knock-out clones.
FAQs
The donor template carries the DNA sequence researchers want cells to incorporate and provides guidance for repair after a targeted break. Cellular repair can use homology-directed repair, in which matching regions help position the incoming sequence, or related mechanisms. This connection between the break and donor template enables specific changes rather than an unspecified genomic alteration.
A defined genomic location links the inserted sequence to a particular gene or regulatory context. That precision allows researchers to examine how a mutation, reporter gene, epitope tag, or human disease variant affects gene function. It also supports interpretations based on the intended locus, rather than changes introduced at an unrelated site.
CRISPR-Cas systems provide the targeted DNA break that initiates the modification, while the cell’s repair machinery determines how the supplied donor sequence becomes incorporated. The two components therefore perform different but connected functions: one directs the editing event to a chosen genomic site, and the other uses the available repair pathway to complete the change.
A typical workflow selects a genomic site, uses a CRISPR-Cas system to create a targeted break, and supplies a donor template containing the desired sequence. Cellular repair then incorporates that sequence through homology-directed repair or a related mechanism. The resulting engineered cells or organisms can be examined for the intended change and its effect on gene function.
Researchers can use the method to introduce mutations, reporter genes, epitope tags, or human disease variants. These payloads serve different experimental purposes: mutations alter gene function, reporter genes help follow gene activity, epitope tags support investigation of a protein, and disease variants model medically relevant genetic changes in engineered systems.
The approach is useful when researchers need a defined genetic change to investigate gene regulation, protein localization, or disease mechanisms. Engineered cells and model organisms can carry reporters, tags, mutations, or disease variants, creating experimental systems that connect a specific DNA alteration with observable biological behavior and that can also inform studies of potential therapeutic strategies.