CRISPR Library Screening to study targeted gene (CAT#: STEM-MB-0030-WXH)

Introduction

CRISPR Screen is a high-throughput screening method of important molecular targets. An efficient CRISPR Screen starts with a well-designed sgRNA library targeting the genes or loci of interest. sgRNA are designed based on sequence and then synthesis followed by cloning to vector or transcribing into RNA for transfection in cell system. Then conduct a screen with broadly used positive or negative selection. PCR amplification and massively parallel sequencing will be performed following physical separation of cells into two or more populations. After data analysis, the sgRNA and targeted genes of interest will be identified.

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Principle Applications Procedure Materials Notes Related Products

Applications

• Determine the relationship between phenotype and genotype
• looking for genes that cause drug resistance or drug sensitivity in cells
• Identify genes important in mitochondrial metabolism and lysosome function
• Turn on expression of thousands of long non-coding RNAs (lncRNAs) to identify those involved in regulating drug resistance in melanoma cells

Procedure

1. Designing synthetic sgRNA libraries
2. construction of corresponding cell lines for stable expression of cas9 protein
3. construction of lentiviral vectors and infection of host cells
4. Positive or negative screening
5. High-throughput sequencing and bioinformatic analysis
Bioinformatics methods are used to find candidate genes and exclude false-positive or false-negative screening results.
6. Validation of candidate genes

Notes

Customer provides cell name, screening index, whole genome or a pathway.

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