WGS-Seq    

CRISPR-Cas9 system is a popular editing tool and widely used in different fields, revolutionizing gene research and biomedicine. However, putative off-target effect is a major concern when using this tool. CRISPR/Cas9 system works by targeting specific DNA sequences to make precise cuts and edits. Since the similarity of sequence, it may inadvertently interact with unintended genomic sites, leading to off-target alterations. These unintended changes may have significant consequences, like disruptions to normal gene function, which could result in terrible side effects or diseases. Thus, it is critical and essential to assess the off-target effects before use.

Whole Genome Sequencing (WGS) is one of the most thorough and objective methods for identifying off-target cleavages genome-wide. By sequencing the whole genome in certain depth, WGS makes it possible to locate predicted and unpredicted off-target regions throughout the genome. WGS provides an unbiased and high-resolution method to detect all off-target modifications.

Creative Biogene's CRISPR Platform^CB provides a WGS-based off-target analysis service, including gDNA extraction, library preparation, sequencing and bioinformatics analysis. With our excellent sequencing platform and experienced bioinformatics team, we provide precise and comprehensive analysis to find all detected off-target sites. With our data, you may investigate all SNP/InDel locations inside sgRNA homologous regions, allowing you to filter and analyze certain areas or genes of interest.

Table 1. Off-target Detection Service Content

Analysis List	Detailed Analysis Content
Variant Screening	SNP/Indel, SV Variant Screening
Off-Target Variant Analysis	Off-Target Variant Screening
	Statistics of Off-Target Homologous sgRNA Regions
	Conservation Plot of Off-Target Homologous Regions
	Off-Target Variant Distribution Map
	On-Target/Off-Target Rate
Annotation	Functional Annotation of Variant Sites in Disease

Applications of Our Off-target Detection Service

1. Standard gene editing off-target detection: Gene knockout, Gene knock-in, Point mutations.

2. Base editing-induced random sgRNA-independent off-target effects

3. Prime editing-induced sgRNA-dependent off-target effects

4. Chromosomal rearrangements and structural variations

Workflow

1. Sample Processing

• DNA extraction and quality control
• Library preparation using standard protocols

2. Sequencing

• High-throughput sequencing on advanced platforms
• 50x coverage depth for accurate variant detection

3. Data Analysis Pipeline

• Raw data processing and quality filtering
• Alignment to the reference genome using BWA
• Variant calling using SAMtools and bcftools
• Advanced filtering (QUAL>20, DP>4, MQ>30)
• Off-target site identification and analysis

Figure 1. WGS Analysis Workflow

4. Project Timeline

Standard service: 25-35 working days

Timeline breakdown:

• Sample QC and library preparation: 7-9 days
• Sequencing: 7-10 days
• Data analysis and report generation: 8-15 days

Sample Requirements

Table 2. Sample Quality for Detection Requirements

DNA Requirements
Quantity	≥2 µg total
Concentration	≥50 ng/µL
Quality	OD260/280 ratio between 1.8-2.0
Integrity	No obvious degradation
Sample Types Required
For Base Editing:
Experimental group	Base editors + sgRNA
Control group	Base editors only
Negative control	Untreated
For Prime Editing and Other Gene Editing:
Experimental group	Editors + sgRNA
Negative control	Untreated

Service Advantages

• Precise Positioning: Our service provides accurate genome-wide identification of both on-target and off-target sites with single-nucleotide resolution.
• Unbiased Analysis: Our platform ensures comprehensive detection of all variant types across the entire genome, eliminating detection bias and enabling the discovery of unexpected modifications.
• Low Background Noise: Advanced sequencing technology and optimized protocols enable the detection of low-frequency variants with high confidence.
• Simple Design: No cell model restrictions, offering flexibility across various experimental designs and applications.
• High Throughput: Updated sequencing platforms accommodate large-scale projects with enhanced throughput capacity.
• Rich Experience: Extensive expertise in handling large population and cohort studies, ensuring reliable and reproducible results.