NDLI: Decoding the complexity of on-target integration: characterizing DNA insertions at the CRISPR-Cas9 targeted locus using nanopore sequencing

Content Provider	Springer Nature : BioMed Central
Author	Zhao, Juan-Juan Sun, Xin-Yu Tian, Sai-Ning Zhao, Zong-Ze Yin, Meng-Di Zhao, Mei Zhang, Feng Li, Si-Ang Yang, Zhi-Xue Wen, Wei Cheng, Tao Gong, An Zhang, Jian-Ping Zhang, Xiao-Bing
Abstract	Background CRISPR-Cas9 technology has advanced in vivo gene therapy for disorders like hemophilia A, notably through the successful targeted incorporation of the F8 gene into the Alb locus in hepatocytes, effectively curing this disorder in mice. However, thoroughly evaluating the safety and specificity of this therapy is essential. Our study introduces a novel methodology to analyze complex insertion sequences at the on-target edited locus, utilizing barcoded long-range PCR, CRISPR RNP-mediated deletion of unedited alleles, magnetic bead-based long amplicon enrichment, and nanopore sequencing. Results We identified the expected F8 insertions and various fragment combinations resulting from the in vivo linearization of the double-cut plasmid donor. Notably, our research is the first to document insertions exceeding ten kbp. We also found that a small proportion of these insertions were derived from sources other than donor plasmids, including Cas9-sgRNA plasmids, genomic DNA fragments, and LINE-1 elements. Conclusions Our study presents a robust method for analyzing the complexity of on-target editing, particularly for in vivo long insertions, where donor template integration can be challenging. This work offers a new tool for quality control in gene editing outcomes and underscores the importance of detailed characterization of edited genomic sequences. Our findings have significant implications for enhancing the safety and effectiveness of CRISPR-Cas9 gene therapy in treating various disorders, including hemophilia A.
Related Links	https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/s12864-024-10050-6.pdf
Ending Page	23
Page Count	23
Starting Page	1
File Format	HTM / HTML
ISSN	14712164
DOI	10.1186/s12864-024-10050-6
Journal	BMC Genomics
Issue Number	1
Volume Number	25
Language	English
Publisher	BioMed Central
Publisher Date	2024-02-17
Access Restriction	Open
Subject Keyword	Life Sciences Microarrays Proteomics Animal Genetics and Genomics Microbial Genetics and Genomics Plant Genetics and Genomics CRISPR-Cas9 Gene therapy DNA integration Long-range PCR Nanopore sequencing
Content Type	Text
Resource Type	Article
Subject	Biotechnology Genetics
Journal Impact Factor	3.5/2023
5-Year Journal Impact Factor	4.1/2023

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
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4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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