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| Rapid on-site genotyping of the ovine prolific FecBB mutation using a CRISPR/Cas12a-based detection system |
| Tingjie Wu1*, Jiayuan Sun1, Lijin Lu1, Chen Wang1, Shiwei Zhou2,3, Yulin Chen1,3,4, Xinjie Wang5# and Xiaolong Wang1,3,4# |
1International Joint Agriculture Research Center for Animal Bio-breeding, Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, China
2College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, China
3Key Laboratory of Livestock Biology, Northwest A&F University, Yangling, 712100, China
4School of Future Technology on Bio-breeding, Northwest A&F University, Yangling, 712100, China
5Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 518124, China
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摘要 长期以来，提升繁殖力是畜禽育种的重要目标。BMPR1B是影响绵羊产羔数性状的关键基因，该基因外显子上第746位A突变为G，形成了FecBB突变。与野生型绵羊相比，携带FecBB突变的绵羊群体产羔率明显更高。因此，开发一种快速、准确、适用多场景的FecBB突变检测方法用于高繁绵羊群体的选育，对促进肉羊产业发展意义重大。本研究将CRISPR/Cas12a系统与重组酶聚合酶扩增（RPA）技术相结合，通过在RPA引物上设计单个错配碱基，形成Cas12a所需的原间隔邻接基序（PAM）序列。随后，采用双crRNA策略，在CRISPR RNA（crRNA）中引入错配碱基，筛选出荧光检测结果可肉眼区分的高信噪比crRNA组合。通过使用核酸释放剂进行DNA样本制备，避免了传统基因组DNA提取的复杂步骤，进一步缩短了检测时间。最终，本研究对来自4个品种（系）的56只绵羊血液样本进行检测，结果与Sanger测序结果高度一致，验证了该方法的准确性。总之，本研究基于CRISPR/Cas12a系统和RPA技术为绵羊FecBB突变基因分型提供了一种快速简便、可现场使用的检测方法，本研究是首个结合RPA技术与CRISPR/Cas12a检测系统应用于动物重要经济性状SNP分型方法，理论上适用于任何SNP位点，该方法有望提高家畜育种中SNP突变检测的效率。
Abstract BMPR1B is a pivotal gene that influences reproductive performance in sheep. The sheep populations that carry the FecBB mutation within this gene exhibit significantly higher lambing rates compared to wild-type populations. Therefore, screening for individuals carrying the FecBB mutation is crucial for effective sheep breeding programs. This study aims to establish a rapid, precise, and visualised on-site detection method for genotyping the prolific FecBB mutation in sheep. We combined the CRISPR/Cas12a system with the recombinase-polymerase amplification (RPA) technique. We introduced an additional nucleotide mismatch on the amplification primers to form a Cas12a-recognised protospacer adjacent motif (PAM) sequence. In addition, mismatches were introduced in CRISPR-derived RNA (crRNA) to enable naked-eye differentiation of the assay results. Subsequently, we validated the accuracy of the method by examining additional blood samples from 56 sheep representing four breeds. The results of using our developed system were highly consistent with the Sanger sequencing. Overall, the CRISPR/Cas12a-based detection provides a rapid and more versatitle method for FecBB genotyping. It holds promise in enhancing efficiency in livestock breeding programmes for any single nucleotide mutations.
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Online: 30 May 2024
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| About author: Tingjie Wu, Tel: +86-13091718973, E-mail: tingjie.wu@nwafu.edu.cn; #Correspondence Xinjie Wang, Tel: +86-29 87091130, E-mail: wangxinjie@caas.cn; Xiaolong Wang, Tel: +86-29-87091130, E-mail: xiaolongwang@nwafu.edu.cn.
*These authors contributed equally to this work. |
Cite this article:
Tingjie Wu, Jiayuan Sun, Lijin Lu, Chen Wang, Shiwei Zhou, Yulin Chen, Xinjie Wang, Xiaolong Wang.
2026.
Rapid on-site genotyping of the ovine prolific FecBB mutation using a CRISPR/Cas12a-based detection system. Journal of Integrative Agriculture, 25(4): 0-.
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