Whole-Genome Sequencing Reveals Rare Off-Target Mutations in MC1R-Edited Pigs Generated by Using CRISPR-Cas9 and Somatic Cell Nuclear Transfer.

IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY CRISPR Journal Pub Date : 2024-02-01 DOI:10.1089/crispr.2023.0034
Zhenyang Li, Jin Lan, Xuan Shi, Tong Lu, Xiaoli Hu, Xiaohong Liu, Yaosheng Chen, Zuyong He
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Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used to create animal models for biomedical and agricultural use owing to its low cost and easy handling. However, the occurrence of erroneous cleavage (off-targeting) may raise certain concerns for the practical application of the CRISPR-Cas9 system. In this study, we created a melanocortin 1 receptor (MC1R)-edited pig model through somatic cell nuclear transfer (SCNT) by using porcine kidney cells modified by the CRISPR-Cas9 system. We then carried out whole-genome sequencing of two MC1R-edited pigs and two cloned wild-type siblings, together with the donor cells, to assess the genome-wide presence of single-nucleotide variants and small insertions and deletions (indels) and found only one candidate off-target indel in both MC1R-edited pigs. In summary, our study indicates that the minimal off-targeting effect induced by CRISPR-Cas9 may not be a major concern in gene-edited pigs created by SCNT.

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全基因组测序揭示了利用 CRISPR-Cas9 和体细胞核移植技术生成的 MC1R 编辑猪的罕见脱靶突变。
聚类规则间隔短回文重复序列(CRISPR)-Cas9 系统因其成本低、操作简便而被广泛用于创建生物医学和农业用途的动物模型。然而,错误切割(脱靶)的发生可能会给 CRISPR-Cas9 系统的实际应用带来一定的隐患。在本研究中,我们利用经CRISPR-Cas9系统修饰的猪肾细胞,通过体细胞核移植(SCNT)创建了黑皮质素1受体(MC1R)编辑猪模型。然后,我们对两头MC1R编辑猪和两头克隆的野生型同胞以及供体细胞进行了全基因组测序,以评估全基因组范围内是否存在单核苷酸变异以及小的插入和缺失(indel),结果在两头MC1R编辑猪中都只发现了一个候选的脱靶indel。总之,我们的研究表明,CRISPR-Cas9 诱导的最小脱靶效应可能不是 SCNT 基因编辑猪的主要问题。
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来源期刊
CRISPR Journal
CRISPR Journal Biochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
6.30
自引率
2.70%
发文量
76
期刊介绍: In recognition of this extraordinary scientific and technological era, Mary Ann Liebert, Inc., publishers recently announced the creation of The CRISPR Journal -- an international, multidisciplinary peer-reviewed journal publishing outstanding research on the myriad applications and underlying technology of CRISPR. Debuting in 2018, The CRISPR Journal will be published online and in print with flexible open access options, providing a high-profile venue for groundbreaking research, as well as lively and provocative commentary, analysis, and debate. The CRISPR Journal adds an exciting and dynamic component to the Mary Ann Liebert, Inc. portfolio, which includes GEN (Genetic Engineering & Biotechnology News) and more than 80 leading peer-reviewed journals.
期刊最新文献
From Code to Comprehension: AI Captures the Language of Life. Response to Cook et al. re: Novel Off-Targeting Events Identified After Genome Wide Analysis of CRISPR-Cas Edited Pig. Managing Expectations for CRISPR in a Volatile World. Efficient Generation of SOCS2 Knock-Out Sheep by Electroporation of CRISPR-Cas9 Ribonucleoprotein Complex with Dual-sgRNAs. An Efficient and Cost-Effective Novel Strategy for Identifying CRISPR-Cas-Mediated Mutants in Plant Offspring.
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