Large Fragment Deletions Induced by Cas9 Cleavage While Not in BEs System in Rabbit

Abstract

CRISPR-Cas9 and BEs system are poised to become the gene editing tool of choice in clinical contexts, however large fragment deletion was found in Cas9-mediated mutation cells without animal level validation. By analyzing 16 gene-edited rabbit lines (including 112 rabbits) generated using SpCas9, BEs, xCas9 and xCas9-BEs with long-range PCR genotyping and long-read sequencing by PacBio platform, we show that extending thousands of bases fragment deletions in single-guide RNA/Cas9 and xCas9 system mutation rabbit, but few large deletions were found in BEs-induced mutation rabbits. We firstly validated that no large fragment deletion induced by BEs system at animal level, suggesting that BE systems can be beneficial tools for the further development of highly accurate and secure gene therapy for the clinical treatment of human genetic disorders.

Funding Statement: This study was financially supported by the National Key Research and Development Program of China Stem Cell and Translational Research (2017YFA0105101). The Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030501, XDA16030503), Guangdong Province Science and Technology Plan Project (2014B020225003). China Postdoctoral Science Foundation Funded Project (2019T120236, 2018M641784).

Declaration of Interests: The authors declare that they have no competing interests.

Ethics Approval Statement: All animal studies were conducted according to the experimental practices and standards approved by the Animal Welfare and Research Ethics Committee at Jilin University.

Keywords: large fragment deletion, CRISPR/Cas9, BEs, Rabbits