CRISPR Screening

Overview
Services
Deliverables
Items to prepare

Inquiry

Overview

Our next-generation sequencing service supports genome-wide screening using CRISPR gRNA libraries. By providing PCR products of the gRNA region, we conduct deep sequencing and perform gRNA read count analysis.

Services

Data Amount

The data amount is calculated as follows:
Read Pair Count x Read Length x 2 (paired-end)

For example, sequencing 10 million read pairs at 150 bp:
10,000,000 x 150 bp x 2 = 3,000,000,000 b = 3 Gb

Coverage is determined by the following formula:
Read Pair Count / Number of gRNA Types = Coverage

For CRISPR screening analysis, coverage of approximately 100 – 500x is generally recommended.

For example, for a library with 120,000 types of gRNA at 100x coverage:
120,000 x 100 = 12 million read pairs, resulting in:
12,000,000 x 150 bp x 2 = 3.6 Gb

Please specify the desired data amount (read pair count) based on the type and desired coverage of your gRNA library.

Deliverables

Item	Content	Format	Details
Reports	・Work Report	Document	Overview of the workflow
Reports	・Sequencing Report	Document	Details of the sequencing data
Media – DVD-R – USB – HDD, etc.	・Sequencing Data	fastq / txt / xlsx	Raw sequence data including corresponding quality scores (fastq) and a summary of sequencing results
	・Preprocessing Data	fastq / txt / xlsx	Data after quality control (QC) and trimming, along with a summary of the pre-processing steps
	・gRNA Count Data	txt / xlsx	gRNA count data for each sample, calculated using the MAGeCK program (Example Data 1)
	・Comparison Table	txt / xlsx	Comparison results of gRNA abundance between samples or groups, analyzed using the MAGeCK program (Example Data 2 / Example Data 3)
	・Reference Data	txt	gRNA library sequence data used in the analysis
	・Parameters	txt	List of programs and parameters used in the analysis
	・Analysis Report	txt	Comprehensive documentation on data analysis
	・Supplementary	txt	Guidance on free software for viewing fastq files and an overview of the library structure

Example of Delivered Data: gRNA Read Count Table

gRNA ID	gRNA Sequence	SampleA Count
ID_0001	AAATACAAACAACTCTGAG	213
ID_0002	GAAGTGTCCCAGGGCGAGG	294
ID_0003	ACTCTATCACATCACACTG	35

Example of Delivered Data: gRNA Count Comparison Table

gRNA ID	Control		Test		LOG2FC	p_value	FDR	High in Treat
gRNA ID	SampleA	SampleB	SampleC	SampleD	LOG2FC	p_value	FDR	High in Treat
ID_0001	213	274	883	175	1.12	0.9996	0.00699	FALSE
ID_0002	294	412	1554	1891	2.29	0.0000	0.00000	TRUE
ID_0003	421	368	566	759	0.75	0.9995	0.00726	FALSE

Example of Delivered Data: Positive/Negative Selection of Target Genes

Gene	negative selection			positive selection
Gene	p_value	FDR	Rank	p_value	FDR	Rank
RPS5	0.000	0.000236	1	1.000	1.000	19147
YARS	0.000	0.000236	2	1.000	1.000	19146
GTF2B	0.000	0.000236	3	1.000	1.000	19145
NRF1	0.000	0.000236	4	1.000	1.000	19144
NLE1	0.000	0.000236	5	1.000	1.000	19143

Items to prepare

Sample

PCR product of the gRNA region

■ PCR Product Structure

1. Prepare PCR products with linkers added using primers with our specified linker sequences (contact us for details).
2. The insert size (excluding linkers) should range between 200-400 bp, with the gRNA region located within the first 100 bp from the 5′ end. Design primers accordingly.
3. Submit PCR products with at least 5 ng (0.2 ng/µL or higher).
4. Purify PCR products to remove primers and nonspecific byproducts.

1st PCR Template Requirements

To ensure comprehensive coverage of sgRNA variations in CRISPR screening experiments, calculate the required template amount for the 1st PCR based on the number of cells used for genome extraction and the sgRNA library.

For example, the genome DNA weight per human or mouse cell is approximately 6.6-7 pg. For an sgRNA library with 50,000 types and 300-500x coverage:The required template DNA is estimated to be approximately 100 µg.

Adjust PCR volumes and the number of reactions based on your screening conditions and sgRNA library specifications.

Control Samples

Control samples are recommended to assess the impact of screening on genome editing.

Examples of control samples include:
Day 0 (untreated) cells post-transfection
The gRNA library used before transfection

Reference Data

gRNA List

Provide the sequence information of the gRNA library. This can include:
Purchase details (URL or catalog number)
A list of IDs, sequences, and target genes

■ Example of gRNA List

gRNA ID	gRNA sequence	Gene
ID_0001	AAATACAAACAACTCTGAG	GeneA
ID_0002	GAAGTGTCCCAGGGCGAGG	GeneB
ID_0003	ACTCTATCACATCACACTG	GeneC

Required Documentation

Order Form

Download the appropriate form, complete the required details, and submit it.
Illumina Order Form (For CRISPR Screening, JPN)

Back to "Products & Services"