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Journal of Integrative Agriculture  2026, Vol. 25 Issue (4): 1597-1605    DOI: 10.1016/j.jia.2024.05.013
Animal Science · Veterinary Medicine Advanced Online Publication | Current Issue | Archive | Adv Search |
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#, Xiaolong Wang1, 3, 4#

1 International 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

2 College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China

3 Key Laboratory of Livestock Biology, Northwest A&F University, Yangling 712100, China

4 School of Future Technology on Bio-breeding, Northwest A&F University, Yangling 712100, China

5 Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518124, China 

 Highlights 
A rapid, precise, on-site, portable, and naked-eye visualization method for genotyping the FecBB mutation in sheep has been established.
A genotyping strategy for important SNP site in livestock breeding programmes is provided, theoretically applicable to any SNP site.
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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746A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,促进肉羊产业发展意义重大本研究CRISPR/Cas12a系统与重组酶聚合酶扩增(RPA)技术相结合,通过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 RNAcrRNA)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)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技术绵羊FecBB突变基因分型提供了一种快速简便、可现场使用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.  

Keywords:  CRISPR/Cas12a       isothermal amplification       SNP detection       BMPR1B       FecBB mutation       sheep  
Received: 20 January 2024   Accepted: 15 March 2024 Online: 15 May 2024  
Fund: This research was supported by the Sci-Tech Innovation 2030 Key Program of China (2022ZD0401401), the National Natural Science Foundation of China (U23A20228, 32272848, 32202646, and 32161143010), the Key Special Project of Ningxia Science and Technology Department, China (2021BEF02024 and 2023BCF01005), the China Agricultural Research System (CARS-39-03), and local grant (2022GD-TSLD-46).  
About author:  Tingjie Wu, E-mail: tingjie.wu@nwafu.edu.cn; #Correspondence Xinjie Wang, E-mail: wangxinjie@caas.cn; Xiaolong Wang, Tel: +86-29-87091130, E-mail: xiaolongwang@nwafu.edu.cn

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): 1597-1605.

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