Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter

作者： Changkun Hu*1, Tyler Doerksen*2, Taylor Bugbee1, Nicholas A. Wallace1, Rachel Palinski2,3 1Division of Biology,Kansas State University, 2Kansas State Veterinary Diagnostic Laboratory,Kansas State University, 3Department of Diagnostic Medicine/Pathobiology,Kansas State University

简介：

Overview

This protocol helps identify mutations that occur during the repair of a double-strand break induced by sgRNA and CAS9. It is a cost-effective and robust method applicable to various transfectable cell lines.

Key Study Components

Area of Science

• Genetic Engineering
• Cell Biology
• Mutation Analysis

Background

• Double-strand breaks are critical events in DNA repair.
• sgRNA/CAS9 technology is widely used for genome editing.
• Identifying mutations can provide insights into repair mechanisms.
• This protocol can be adapted for different cell types.

Purpose of Study

• To develop a protocol for identifying mutations at specific genomic loci.
• To demonstrate the effectiveness of sgRNA/CAS9 in mutation detection.
• To provide a reliable method for researchers in genetic studies.

Methods Used

• Culture human keratinocyte cells in specific conditions.
• Use trypsin-EDTA for cell detachment.
• Apply sgRNA/CAS9 for inducing double-strand breaks.
• Perform next-generation sequencing for mutation analysis.

Main Results

• Successful identification of mutations associated with DNA repair.
• Demonstrated robustness of the protocol across cell lines.
• Cost-effective approach for mutation detection.
• Potential for broader applications in genetic research.

Conclusions

• The sgRNA/CAS9 protocol is effective for mutation analysis.
• Adaptable to various transfectable cell lines.
• Provides a valuable tool for researchers studying DNA repair mechanisms.

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