Grepore-seq: A Robust Workflow for Detecting Gene Editing Changes through Long-Range PCR and Nanopore Sequencing

Research Background

The CRISPR/Cas9 system, as an RNA-guided DNA endonuclease system, has been widely applied in genome editing. As its potential in clinical treatment continues to increase, comprehensive evaluation of gene editing results has become particularly important. However, there is currently a lack of a large-scale, cost-effective, and pipeline-like method to detect the results of gene editing, especially in cases of large fragment insertions or deletions. Research has shown that unintended effects may occur after CRISPR/Cas9 editing, such as large fragment deletions and complex genomic rearrangements, which pose challenges to its clinical application.

Research Objectives and Innovation

To address the above issues, this study introduces a new processing workflow called “grepore-seq”, which combines long-range PCR and nanopore sequencing to efficiently and accurately detect various gene changes after CRISPR/Cas9 editing. Due to the high error rate of Oxford Nanopore sequencing, researchers developed a novel pipeline that captures barcoded sequences by grepping the read sequences of nanopore amplicons. This workflow, named Grepsseq, can evaluate various gene changes after CRISPR/Cas9 editing, such as NHEJ-mediated dsODN insertion and HDR-mediated large fragment insertion, and shows good consistency with Illumina next-generation sequencing results.

Research Source

This study was jointly conducted by Zi-Jun Quan, Si-Ang Li, Zhi-Xue Yang, Juan-Juan Zhao, and others, mainly from the research team at the Institute of Hematology, Chinese Academy of Medical Sciences. The paper was published in the journal “Genomics Proteomics Bioinformatics” in 2023.

Research Process

Research Subjects and Sample Processing

The study first performed gene editing on K562 cells, human T cells, hematopoietic stem/progenitor cells, and induced pluripotent stem cells. RNP editors were introduced into these cells through electroporation. Three to four days later, genomic DNA was extracted and long-range PCR was performed to capture 4-8 kb fragments around the guide RNA target sites. Barcoded primers were added to the 5’ end of the forward primer to facilitate pooled sequencing of long amplicons.

Experimental Part and Data Analysis

1. PCR Optimization: By comparing KAPA HiFi DNA polymerase, NileHifi long amplicon PCR kit, and PrimeSTAR GXL DNA polymerase, the study found that PrimeSTAR performed best in terms of specificity and yield. Therefore, PrimeSTAR was used for long-range PCR in subsequent experiments.
2. Data Processing and Analysis: Guppy was used for initial data processing and adapter trimming. Then, Porechop was used to trim the end sequences, and Minimap2 was used to align the read sequences with the reference sequences. Finally, the generated BAM files were visualized using IGV, and self-developed scripts were used to analyze gene editing results after dsODN insertion, HDR insertion, plasmid backbone insertion, or large fragment deletion.

Data Reliability Verification

The study compared the results of Illumina sequencing and grepore-seq to verify the accuracy of grepore-seq in detecting short fragment insertions. The results showed that grepore-seq could effectively and accurately detect 29 bp dsODN insertions and HDR-mediated large fragment insertions, and showed high correlation with Illumina sequencing results.

Research Results

Main Findings

1. Effective Extraction of Long-Range PCR Read Sequences: The study optimized PCR conditions and successfully amplified 4-8 kb fragments using PrimeSTAR GXL DNA polymerase.
2. Integrity and Accuracy of Read Sequences: Through data trimming processing combining Guppy and Porechop, the study found that the length distribution of processed read sequences was more concentrated, reducing the error rate.
3. Barcode Demultiplexing Effect: Grepore-seq showed a higher data recovery rate and lower false discovery rate compared to Barcode_splitter when processing long amplicons with multiple barcodes.
4. Detection of HDR-Mediated Large Fragment Insertions: Using double-cut donor plasmids overlapping the CRISPR target site for HDR editing, grepore-seq successfully detected longer HDR insertions and showed excellent linear correlation with FACS analysis results.
5. Detection of Plasmid Backbone Insertions: The study revealed a certain proportion of plasmid backbone insertions and verified the role of the NHEJ pathway in plasmid backbone insertion through NHEJ inhibition experiments.

Research Conclusions and Significance

1. Scientific Significance: This study established an efficient and accurate workflow for evaluating various genomic changes after CRISPR/Cas9 editing. Grepore-seq not only performs excellently in data processing and analysis but can also effectively address large fragment insertions and deletions that are difficult to detect with existing technologies.
2. Application Value: This workflow has economic and scalability advantages and can be applied to rapid, large-scale assessment of various genomic changes after gene editing. This provides a new technical means for gene editing research and clinical applications.

Research Highlights

1. Workflow Innovation: Combining long-range PCR with nanopore sequencing, a new data processing pipeline, grepore-seq, was developed.
2. Data Accuracy: Through various optimization schemes and steps, high precision and low error rates of data are ensured.
3. Comprehensiveness: Can detect various CRISPR/Cas9-mediated gene changes, including short fragment insertions, large fragment insertions, plasmid backbone insertions, and large fragment deletions.

Conclusion

Grepore-seq provides a robust and efficient method for detecting gene changes after CRISPR/Cas9 gene editing. This workflow is not only suitable for scientific experiments but also lays a technical foundation for future clinical applications.