Cas9-induced Mutations
Cas9-induced Mutations - Related Videos
10:07
A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells
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Cited by 5 •
2017
This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site—in essence, analyzing onsite mutagenesis.
01:30
Spontaneous and Induced Mutations
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2025
Spontaneous mutations arise infrequently during DNA replication due to errors in the process. A key factor behind these errors is tautomeric shifts in nitrogenous bases, where bases transition from keto to enol forms or amino to imino forms. This shift can alter base-pairing rules, leading to mutations. Additionally, reactive oxygen species (ROS) arising from aerobic metabolism can damage DNA, resulting in depurination (loss of a purine base) or depyrimidination (loss of a pyrimidine base).
06:59
Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter
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Cited by 5 •
2022
Presented here is sgRNA/CAS9 endonuclease and next-generation sequencing protocol that can be used to identify the mutations associated with double strand break repair near the CD4 promoter.
02:36
Establishing a Murine Tumor Cell Line In Vitro Using a Virus-Cas9 System
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2026
Source: Prasad, M. et al. In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System. J. Vis. Exp. (2020)This video demonstrates the in vitro transformation of murine epithelial cells through viral delivery of Cre recombinase and mutation-inducing components. It outlines the steps for Cas9 activation, cancer-gene modification, and the establishment of a tumorigenic murine cell line.
07:45
Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9
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Cited by 5 •
2020
CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.
FAQs
The guide RNA provides sequence complementarity that directs Cas9 toward a matching DNA region, but binding and cutting also require a nearby protospacer adjacent motif, or PAM. This means a potential target is defined by both its sequence match to the guide and the presence of the appropriate neighboring motif. Together, these features constrain where mutations can be generated.
The cellular repair pathway largely determines the type of sequence change produced after Cas9 cutting. Non-homologous end joining can reconnect the broken DNA while introducing insertions or deletions, creating disruptive mutations. Template-directed repair instead uses a repair template to install a defined sequence change, making it suitable when a specific genetic alteration is required.
These outcomes arise from the contrast between imprecise and template-guided repair. Insertions or deletions generated during non-homologous end joining can disrupt a gene and support knockout analysis. When template-directed repair is used, researchers can introduce a defined sequence change instead, allowing a more specific alteration for functional or genetic studies.
Researchers must identify a DNA region that matches the intended guide RNA sequence and lies near a suitable protospacer adjacent motif. That target arrangement determines whether Cas9 can be directed to the selected locus. The planned outcome also matters: non-homologous end joining supports insertion or deletion formation, whereas template-directed repair supports a defined change.
They are useful when researchers need to connect a gene or sequence change with a biological function. Gene knockout studies can examine the consequences of disrupting a locus, while targeted sequence changes can support functional analysis and genetic variation studies. The same approach also contributes to disease modeling and the development of engineered organisms.
By altering a selected genetic region and examining the resulting consequences, researchers can study how genotype relates to phenotype. Cas9-induced mutations therefore provide a framework for testing gene function rather than only observing naturally occurring variation. In genetics, this supports functional analysis, disease models, and studies of how particular sequence changes contribute to biological outcomes.