Long-Read-Seq    

One ongoing concern with CRISPR-Cas9 genome editing is off-target mutations induced by unexpected sgRNA binding. CRISPR-Cas9 works by recognizing specific DNA sequences and inducing targeted cuts. However, due to sequence similarities, unintended genomic sites may also be edited, leading to off-target mutations which may disrupt normal gene function.

Creative Biogene provides off-target analysis services with over 10 years of gene editing experience. Our Long-read sequencing based on Pacific Biosciences' SMRT technology provides an advanced solution for off-target effect assessment, ensuring accuracy and reliability. Long-read sequencing can detect predicted and unpredicted off-target sites, overcoming the limitations of short-read sequencing methods. Directly observing large DNA regions with single-molecule resolution eliminates PCR amplification bias and captures a comprehensive view of genomic variations. Its exceptional sequencing accuracy, reaching Q40 levels, significantly reduces false positives.

Figure 1. Overview of experiment to examine genome editing at on- and off-target sites in cells. (Höijer I, et al., 2020)

Our long-read-Seq services process includes sample preparation, library preparation, Cas9 digestion and enrichment, and rapid SMRT sequencing for accurate and dependable findings. We will help the researchers find off-target locations, particularly those in "dark" genomic areas inaccessible to short-read sequencing, for a more complete and accurate examination of possible off-target impacts.

Table 1. Sample Preparation Requirements and Quality

Sample Preparation	Requirements
Sample Type	Fresh tissue/cells
	High molecular weight genomic DNA
Sample Quantity	Tissue: ≥100 mg
	Cells: ≥5 × 10⁶ cells
	gDNA: ≥5 μg
Quality Control Indicators	DNA Integrity: >50 kb
	Purity: A260/280 = 1.8-2.0
	Concentration: >50 ng/μl

Detailed Workflow

1. Sample Preparation: Begin with a thorough quality assessment and optimization of DNA extraction, followed by comprehensive verification of sample integrity.

2. Library Preparation: Optimize DNA fragmentation and size selection, then construct the SMRT-bell library, ensuring rigorous quality control throughout the process.

3. Cas9 Digestion and Enrichment: Efficiently form gRNA complexes and enrich them, followed by a stringent purification process to ensure high specificity.

4. Sequencing and Analysis: Perform SMRT Cell sequencing using optimized parameters, with real-time monitoring and ongoing quality control. Finally, conduct a comprehensive bioinformatics analysis to interpret the results accurately.

Figure 2. Overview of the SMRT-OTS (Off-Target Sites) Process: From DNA Sample to PacBio Sequencing. (Höijer I, et al., 2020)

Deliverables

Raw Data Package: Includes SMRT Cell metrics, base-called data in BAM format, CCS reads in FASTQ format, and quality metrics reports.

Visualization Package: Provides IGV session files, custom genome browser tracks, publication-ready figures, and interactive visualizations.

Related Services

CRISPR Off-Target Effects Analysis

PEM-Seq

SgRNA Design and Confirmation

Base Editing by CRISPR