Detect and accurately measure the efficiency of both on- and off-target gene editing
Gene editing approaches have become an important tool in research and the development of potential novel therapeutics.
CRISPR-Cas9, base editing, prime editing, transposons and more have great potential to alter genomes in a controlled way. However, each of these methods can have inaccuracies that may pose potential safety risks if introduced in humans. Some of these unintended outcomes include larger structural variants, and insertions or deletions, or concatemers of inserts, that could be missed by using short-read sequencing methods.
HiFi sequencing allows you to confidently assess those larger variants as well as single base changes to determine
- On- and off-target editing effects
- Insertional mutagenesis
- The potential safety of resulting constructs
Application brief
Highly accurate HiFi reads for gene editing research
With highly accurate long reads (HiFi reads), powered by Single Molecule, Real-Time (SMRT) sequencing technology, you can more comprehensively validate gene editing techniques such as CRISPR-Cas9 approaches.
Use a variety of approaches such as amplicon-based sequencing, amplification-free targeted sequencing, and whole genome sequencing to quantify genome editing outcomes, assess insertion sites and understand the effects of haplotypes and SNVs on genome editing.
HiFi sequencing allows you to assess gene editing in a variety of ways
Use the method that is most appropriate for your research question. Gene editing outcomes can be assessed in a targeted or unbiased manner. Whether your approach would benefit most from targeted amplicon-based, amplification-free or unbiased whole genome sequencing, HiFi enables your work with exceptional data accuracy and allows you to see what you may not see with short-read methods.
Amplicon-based sequencing
SMRT DNA sequencing provides a simple, rapid, quantitative, and sensitive strategy for measuring genome-editing outcomes
Amplification-free sequencing
Avoid the introduction of PCR biases: analyze ON and OFF-target edits. The no-amp protocol uses Cas9 target enrichment to confirm editing outcomes
WGS, an unbiased approach
HiFi whole genome long-read sequencing overcomes limitations of detecting certain variants that short-read sequencing poses, such as structural variants, GC-rich and repetitive regions
Amplicon-based sequencing
Sequence beyond your target with long-range amplicons to fully analyze and quantify the potential outcomes of gene editing approaches. HiFi sequencing allows you to detect genome modifications not visible by short-read sequencing, such as
- Large scale deletions and insertions
- Structural changes
- Complex rearrangements
“SMRT DNA sequencing provides a simple, rapid, quantitative, and sensitive strategy for measuring genome-editing outcomes” *
Assess insertions in greater detail: amplicon-based sequencing allows the more comprehensive characterization of construct integrations at target loci. Assess insertion outcomes such as perfect HDR vs imperfect HDR incl. imperfect, complex and concatemeric integrations.**
** Tei C, et al (2023) Comparable analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous tagging. BioRxiv
Amplification-free sequencing
Enriches for target without amplification
- Avoids PCR biases
- Works in regions that are hard to amplify
Enables the analysis of all on- and off-target editing outcomes and integrations
- Allele-specific resolution
- Overcomes short-read limitations: allows to identify long indels, concatemers, inversions, etc.
“Single-molecule sequencing affords better resolution of editing outcomes after dual gRNA excisions of the repeat region than Sanger sequencing does” *
WGS, an unbiased approach
Off target edits are a potential source of risk and need to be carefully evaluated as part of the development of a novel potential gene editing approach.
Regulatory guidelines suggest assessing the off-target effects of gene editing using multiple modalities, including a targeted as well as an unbiased genome-wide method in conjunction.
HiFi whole genome long-read sequencing overcomes limitations of detecting certain variants that short-read sequencing poses, such as structural variants, GC-rich and repetitive regions. With Sanger-level accuracy and read lengths of up to 20 kb, HiFi sequencing allows you to more comprehensively capture the effects of gene editing.
Additionally, HiFi sequencing provides data at haplotype resolution, allowing you to determine the allele-specific outcomes of your editing approach.
Brochure
HiFi solutions for cell and gene therapy research
Explore how HiFi sequencing can be used to support your gene and cell therapy research
See HiFi sequencing for gene editing in action:
Publication
CRISPR-Cas9 editing to ALS repeat expansions assessed with amplification-free targeted HiFi sequencing
A repeat expansion in the C9orf72 gene is one of the most common known causes of amyotrophic lateral sclerosis (ALS) and Frontotemporal dementia (FTD). The repeat length is associated with disease age of onset and severity.
Contracting the repeat length through the use of CRISPR-Cas9 gene editing could be an attractive potential future treatment. It is therefore important to accurately measure the efficiency of C9orf72 repeat length editing.
The traditional methods such as PCR or Southern blot to assess the repeat length have limitations that single-molecule long-read sequencing can overcome.
This study used amplification-free HiFi sequencing to measure repeat length before and after editing with CRISPR-Cas9 and validated this assay to be applicable to discovery research and beyond.
“…we recommend single-molecule sequencing as the gold standard for verifying unedited and edited cell lines, since it can accurately size the repeat expansion, report on clonality, phase surrounding polymorphisms and determine editing outcomes”*
Publication
Long-read amplicon sequencing reveals various patterns of imprecise repair in CRISPR-Cas-mediated knock-in
Insertion of constructs at target sites through CRISPR-Cas9 induced double-strand breaks followed by homology-directed repair is a powerful research approach as well as a potential strategy to treat certain diseases.
While this is a promising approach, there is increasing evidence that these insertions are not always accurate and can result in a number of undesired outcomes that may be classified as blunt, imperfect, concatenated, and complex.
Long-read amplicon sequencing is a straightforward and powerful approach to measure and quantify the accuracy of gene editing and conduct insertion site analysis. The long read length of HiFi sequencing allows researchers to identify variants that may not be visible with short-read methods, while also providing single base accuracy to detect small variants.
Blog
Diversity of unexpected CRISPR-Cas9 edits revealed by SMRT sequencing
Researchers discover CRISPR-Cas9 method results in far more genomic changes than previously thought — including big deletions and rearrangements — was made possible by the use of long-read SMRT sequencing.
Video
Accurately confirm CRISPR-Cas9 off-target sequencing and analysis
Ida Hoijer of Uppsala University shares her research on developing a long-read sequencing-based method for detection of CRISPR-Cas9 off-target effects, along with a customized analysis pipeline.