CIRCLE-Seq for Interrogation of Off-Target Gene Editing
November 1st, 2024
A significant barrier to technologies like CRISPR is the off-target events that can disrupt vital genes. 'Circularization for In Vitro Reporting of Cleavage Effects by Sequencing' (CIRCLE-seq) is a technique designed to identify unintended cleavage sites. This method maps the genome-wide activity of CRISPR-Cas9 with high sensitivity and without bias.
Our team is dedicated to developing induced pluripotent stem cell, iPSC therapies, for severe, currently incurable skin diseases such as recessive dystrophic epidermolysis bullosa. We aim to determine if precise gene editing coupled with iPSC's differentiation into skin cells can provide a viable treatment option for these conditions. Gene editing in somatic cells and iPSCs using CRISPR-Cas9 technology is transforming regenerative medicine by enabling personalized therapies for various diseases, including genetic skin disorders.
Current challenges in genetic engineering include off-target effects of gene editors. The therapeutic requires gene editors that precisely correct the gene of interest without altering any other locations in the genome. Our challenge is to demonstrate that gene editors do not introduce unintended edits beyond the target gene.
The CIRCLE-seq protocol enables the identification of bonafide potential off-target sites that other similar techniques might miss. A comprehensive analysis of these off-target sites provides valuable insight into the activity of genome editors, enhancing the safety of gene therapies, including our IPSC-based therapy for skin diseases. After growing induced pluripotent stem cells for five days, collect the cells by centrifugation and resuspend them in 10 milliliters of PBS.
Mix six microliters of cell suspension in six microliters of trypan blue for cell counting. After counting, aliquot two times 10 to the power of seven cells per tube. Centrifuge the cells at 300 G for three minutes at 25 degrees Celsius and discard the supernatant.
Prepare the focused ultrasonicator by homing the control arm. Fill the reservoir with purified, deionized water. On the control station laptop, access waterworks and click fill to start filling the system.
Adjust the temperature to 4.5 degrees Celsius. Transfer 25 micrograms of genomic DNA isolated from induced pluripotent stem cells into a microtube. Fill the tube to a total volume of 130 microliters with TE buffer.
Set the conditions to shear the DNA to an average length of 300 base pairs, duration of 10 seconds, peak power of 70, duty factor percent to 20, and cycles burst to 50. Divide the sheared genomic DNA into two portions of 65 microliters each. Purify the samples using 1.8 times the volume of XP beads, followed by magnetic rack separation.
Transfer the supernatant to a new PCR plate and measure the DNA quantity using a spectrophotometer. Finally, run one microliter of the alluded shear genomic DNA on a tape station. To begin, resuspend oSQT1288 and the hairpin adapter to a final concentration of 100 micromolar in TE buffer.
For adapter annealing, mix 40 microliters of oSQT1288, 10 microliters of 10X STE, and 50 microliters of nuclease-free water to reach a total volume of 100 microliters. After adapter annealing, mix 10 microliters of 5X ligation buffer, five microliters of DNA ligase, and five microliters of annealed hairpin adapter, Pipette 20 microliters of the adapter ligation master mix into each eluted DNA specimen containing beads. Place the samples in a thermocycler at 20 degrees Celsius for one hour, then hold at four degrees Celsius indefinitely.
Next, transfer 50 microliters of PEG/NaCl spray solution to the adapter-ligated DNA. Purify the samples using XP beads and magnetic rack separation. Elute the samples with 30 microliters of TE buffer pH eight and decant the supernatants into a new semi-skirted PCR plate.
Combine the samples and quantify the DNA using the dsDNA BR assay. To prepare the circularization master mix, combine eight microliters of nuclease-free water, 10 microliters of 10X TD4 DNA ligase buffer, and two microliters of T4 DNA ligase for a total volume of 20 microliters. Pipette 20 microliters of the circularization master mix to 80 microliters of 500 nanograms of DNA treated with user PNK.
Incubate the sample in a thermocycler at 16 degrees Celsius for 16 hours. The next day, add 100 microliters of XP beads to the circularized DNA and purify the samples, as demonstrated previously. Elute the sample with 38 microliters of TE buffer and decant the supernatant into a new semi-skirted PCR plate.
To cleave purified circularized genomic DNA, prepare in vitro cleavage master mix by combining five microliters of 10X Cas9 buffer, 4.5 microliters of Streptococcus pyogenes Cas9, and 1.5 microliters of genomic RNA for a total volume of 11 microliters. Incubate the cleavage master mix at room temperature for 10 minutes. to form the Cas9 gRNA RNP complexes.
Dilute 125 nanograms of plasmid-safe DNase-treated genomic DNA to a final volume of 39 microliters in nuclease-free water. Then, add 11 microliters of the cleavage master mix to the 39 microliters of DNA for a total volume of 50 microliters. Incubate the mixture in a thermocycler for one hour at 37 degrees Celsius and maintain it at four degrees Celsius indefinitely.
After incubation, add 50 microliters of XP beads to the in vitro cleaved DNA and purify the DNA on a magnetic rack. Elute the purified DNA in 42 microliters of TE buffer. For the analysis of next-generation sequencing data, install Python version 2.7, Burrows-Wheeler Aligner, and SAMtools.
Download the reference genome from the given website. Define the reference genome FASTA file, the output directory for the analysis, and the paths to the BWA and SAMtools commands. Specify the target sequences and the paths to the demultiplexed FASTQ files for both the nuclease-cleaved and control samples.
Execute the standard reference-based analysis using the following command. When executing the full pipeline, locate the output results of each step in a distinct output folder designated for that specific step.
Overview
This article discusses the CIRCLE-seq technique, which identifies off-target cleavage sites in CRISPR-Cas9 gene editing. The method enhances the safety of gene therapies by mapping unintended cleavage events with high sensitivity.
Key Study Components
Area of Science
- Gene editing
- CRISPR-Cas9 technology
- Regenerative medicine
Background
- Induced pluripotent stem cells (iPSCs) are being explored for treating severe skin diseases.
- Off-target effects in gene editing pose significant challenges.
- CIRCLE-seq provides a method to identify potential off-target sites.
- Understanding off-target effects is crucial for the safety of gene therapies.
Purpose of Study
- To develop iPSC therapies for genetic skin disorders.
- To ensure precise gene editing without unintended genome alterations.
- To enhance the safety of CRISPR-based therapies.
Methods Used
- Isolation and preparation of genomic DNA from iPSCs.
- Application of the CIRCLE-seq protocol to identify off-target sites.
- Next-generation sequencing for comprehensive analysis.
- Data analysis using bioinformatics tools like Python and SAMtools.
Main Results
- CIRCLE-seq effectively identifies off-target cleavage events.
- Comprehensive analysis provides insights into genome editing safety.
- Demonstrates the potential of iPSC therapies for skin diseases.
- Highlights the importance of precise gene editing in regenerative medicine.
Conclusions
- CIRCLE-seq is a valuable tool for enhancing the safety of CRISPR technologies.
- Understanding off-target effects is essential for developing effective gene therapies.
- The study supports the advancement of personalized medicine through iPSC applications.
Frequently Asked Questions
Executive Industry Relevance
CIRCLE-seq enables biopharma R&D teams to rigorously interrogate off-target effects of CRISPR-Cas9 gene editing, a critical inflection point for advancing gene therapies. By providing high-sensitivity, genome-wide detection of unintended cleavage sites, CIRCLE-seq supports predictive confidence and mechanistic de-risking in early discovery and translational workflows. This capability is essential for portfolio decisions in regenerative medicine and cell therapy development.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables comprehensive mapping of CRISPR-Cas9 off-target activity for functional target validation.
- Supports mechanistic de-risking by identifying unintended genomic alterations before preclinical advancement.
- Facilitates predictive confidence in gene editing specificity, informing go/no-go decisions.
Screening & Assay Development
- Provides validated genomic DNA substrates for downstream assay development and screening workflows.
- Delivers quantitative, reproducible readouts of cleavage events for assay standardization.
- Enables scalable, high-throughput assessment of guide RNA specificity across multiple targets.
Translational & Preclinical Research
- Aligns off-target profiling with disease-relevant iPSC models for translational continuity.
- Supports risk-adjusted advancement of gene-edited cell therapies by confirming genomic integrity.
- Enhances biomarker alignment by linking cleavage profiles to functional outcomes in preclinical models.
Pipeline & Workflow Integration
CIRCLE-seq is positioned at the intersection of early discovery, lead identification, and preclinical validation, enabling seamless integration into gene editing pipelines.
- Discovery Biology: Supports hypothesis testing and pathway clarification by revealing off-target effects of CRISPR-Cas9.
- Screening: Provides reproducible, quantitative outputs for guide RNA and Cas9 specificity assessment.
- Analytics: Generates high-resolution cleavage site data for robust statistical comparison of editing conditions.
- Translational Research: Bridges discovery and preclinical validation by confirming editing fidelity in disease-relevant systems.
- Enterprise Reuse: Establishes a standardized, scalable workflow for ongoing gene editing safety assessment across programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in gene editing projects.
- Operational Value: Delivers standardized, reproducible, and scalable off-target analysis for cross-team adoption.
- Strategic Value: Enables informed go/no-go decisions and capital-efficient portfolio advancement by de-risking editing specificity.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of gene and cell therapy candidates.
Implementation Considerations
- Requires expertise in genomic DNA preparation, next-generation sequencing, and bioinformatics analysis.
- Demands access to focused ultrasonication, Illumina sequencing platforms, and computational infrastructure.
- Necessitates cross-team standardization of sample preparation and data analysis pipelines.
- Adaptable to various cell types and gene editing targets with protocol optimization.
- Dependent on sequencing quality and reference genome accuracy for reliable off-target detection.
Biopharma FAQs
Chapters in this video
0:00
Introduction
1:23
Cell Culture and DNA Shearing Protocol for Genomic DNA Preparation from Induced Pluripotent Stem Cells
3:28
CIRCLE-Seq Library Preparation for Genomic DNA Circularization and Purification
5:58
In Vitro Cleavage of Circularized Genomic DNA and CIRCLE-Seq Data Analysis
