CRISPR高通量筛选技术及其在畜牧研究中的应用

doi:10.13560/j.cnki.biotech.bull.1985.2025-0500

摘要/Abstract

摘要：

畜牧业是农业的重要组成部分，其经济价值与畜禽表型性状（如产肉量、产蛋率等）密切相关。这些性状由复杂的遗传网络调控，并且受到环境因素的影响，导致挖掘决定这些性状的功能基因较为困难。遗传筛选技术的出现使基因型-表型关系的发现变得简单，但早期的遗传筛选技术通量低并且难以分析产生的大量数据致使效率低下。随着生物技术的迭代更新，遗传筛选技术进入高通量筛选时代，为解析基因型-表型关系提供了高效手段。传统的高通量筛选方法主要包括RNA干扰（RNAi）和cDNA过表达等，但RNAi易脱靶且无法完全抑制基因表达，cDNA文库构建成本高且可能引发细胞毒性。CRISPR/Cas系统的开发促使为遗传筛选提供了新的策略。该系统基于细菌的适应性免疫机制，通过向导RNA靶向DNA序列并利用Cas蛋白精准编辑基因，具有操作简便、特异性高和适用范围广的优势。CRISPR高通量筛选技术主要包括功能缺失型（CRISPRko）、功能获得型（CRISPRa/CRISPRi）、碱基编辑等多种模式，能够直接在DNA水平进行扰动，克服了传统方法的不足。本文介绍了遗传筛选技术的类型、特点以及在畜牧学中的应用，并展望了CRISPR高通量筛选技术在畜牧业育种中的巨大潜力。

关键词: 畜牧业, 遗传筛选, CRISPR高通量筛选, SgRNA文库

Abstract:

Animal husbandry is an important part of agriculture, and its economic value is closely related to the phenotypic traits of livestock and poultry (such as meat production, egg production rate, etc.). These traits are regulated by complex genetic networks and are affected by environmental factors, causing it difficult to mine functional genes that determine these traits. The emergence of genetic screening technology makes the discovery of genotype-phenotype relationship simple, but the early genetic screening technology has low throughput and is difficult to analyze the large amount of data generated, resulting in low efficiency. With the iterative update of biotechnology, genetic screening technology has entered the era of high-throughput screening, which provides an efficient means for analyzing genotype-phenotype relationship. Traditional high-throughput screening methods mainly include RNA interference (RNAi) and cDNA overexpression. However, RNAi is prone to have off-target and cannot completely inhibit gene expression. The construction of cDNA library is costly and may cause cytotoxicity. The development of CRISPR/Cas system provides a new strategy for genetic screening. Based on the adaptive immune mechanism of bacteria, the system targets DNA sequences by guide RNA and accurately edits genes by Cas protein, which has the advantages of simple operation, high specificity and wide application range. CRISPR high-throughput screening technology mainly includes multiple modes such as loss-of-function (CRISPRko), gain-of-function (CRISPRa/CRISPRi), and base editing, which can directly perturb at the DNA level and overcome the shortcomings of traditional methods. This paper briefly introduces the types and characteristics of genetic screening technology and its application in animal husbandry, and looks forward to the great potential of CRISPR high-throughput screening technology in animal husbandry breeding.

Key words: animal husbandry, genetic screening, CRISPR high-throughput screening, SgRNA library

田云, 孔辰, 杨冲, 刘统高, 高红瑞, 李佳慧, 马云, 蔡蓓. CRISPR高通量筛选技术及其在畜牧研究中的应用[J]. 生物技术通报, 2025, 41(12): 82-94.

TIAN Yun, KONG Chen, YANG Chong, LIU Tong-gao, GAO Hong-rui, LI Jia-hui, MA Yun, CAI Bei. CRISPR-based High-throughput Screening Technology and Its Applications in Livestock Research[J]. Biotechnology Bulletin, 2025, 41(12): 82-94.

图/表 6

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特征 Feature	全基因组sgRNA文库 Genome-wide sgRNA library	单一基因的sgRNA Single gene sgRNA
目标范围	全基因组范围（包含数万的基因）	单一或少数基因
sgRNA数量级	数万或数十万条sgRNA	4-10条sgRNA
设计深度	每个基因4-6条sgRNA	每个基因可设计数10条sgRNA
覆盖范围	广度优先（尽可能的覆盖多的基因）	深度优先（聚焦特定基因）
应用场景	未知基因的挖掘	基因功能的精细验证

特征 Feature	全基因组sgRNA文库 Genome-wide sgRNA library	单一基因的sgRNA Single gene sgRNA
目标范围	全基因组范围（包含数万的基因）	单一或少数基因
sgRNA数量级	数万或数十万条sgRNA	4-10条sgRNA
设计深度	每个基因4-6条sgRNA	每个基因可设计数10条sgRNA
覆盖范围	广度优先（尽可能的覆盖多的基因）	深度优先（聚焦特定基因）
应用场景	未知基因的挖掘	基因功能的精细验证

sgRNA文库类型 Type of sgRNA library	sgRNA位点要求 Requirements of sgRNA site	位点要求的原因及目的 Reason and purpose of site requirements
全基因组文库	一般位于编码序列的前25%	确保蛋白功能的完全丧失
激活文库	TSS上游200 bp左右	确保激活结构域能够靶向TSS位点，以实现基因表达的增强
干扰文库	TSS下游+25-75 nts左右	确保转录抑制结构域能够靶向TSS位点，以实现沉默基因
敲除文库	选择编码区前50%（靠近ATG），避开内含子剪切位点	确保蛋白功能的丧失，避免截留的蛋白残基仍存有功能
STOP文库	TSS下游100 bp左右	确保终止密码子的提前引入，以丧失沉默基因。

sgRNA文库类型 Type of sgRNA library	sgRNA位点要求 Requirements of sgRNA site	位点要求的原因及目的 Reason and purpose of site requirements
全基因组文库	一般位于编码序列的前25%	确保蛋白功能的完全丧失
激活文库	TSS上游200 bp左右	确保激活结构域能够靶向TSS位点，以实现基因表达的增强
干扰文库	TSS下游+25-75 nts左右	确保转录抑制结构域能够靶向TSS位点，以实现沉默基因
敲除文库	选择编码区前50%（靠近ATG），避开内含子剪切位点	确保蛋白功能的丧失，避免截留的蛋白残基仍存有功能
STOP文库	TSS下游100 bp左右	确保终止密码子的提前引入，以丧失沉默基因。

筛选工具 Screening tool	扰动方式 Disturbed mode	优点 Merit	局限性 Limitation
RNAi	通过降解mRNA抑制基因表达	简化了筛选方法，降低筛选文库的构建成本	无法针对基因组基因进行筛选；基因沉默不完全，只能在细胞质中行使功能
cDNA/ORF	提高目标蛋白的表达	第一个功能获得型筛选方法，是发现冗余基因功能的方法	文库构建困难，并且成本高；某些基因过表达后会导致细胞死亡
CRISPRko	利用gRNA引导Cas9酶切割目标DNA序列，通过细胞的DNA修复机制导致目标基因敲除	可以最大程度地避免基因的假沉默；可实现非编码基因的筛选	存在脱靶效应；断裂DNA双链引起的DNA毒性；无法研究基因恢复后的情况
CRISPRi	通过阻断转录起始实现基因沉默	不引起DNA双链断裂，造成的损伤是可逆的，脱靶效应小，特异性强	对基因结构有依赖性
CRISPRa	激活转录因子，增强基因转录活性	不引起DNA双链断裂；造成的损伤是可逆的	不能针对特定转录本激活；激活效率受限
BE	将脱氨酶与dCas9结合，实现碱基的转换，从而实现起始密码子和终止密码子的沉默和提前终止，以实现基因的沉默	不引起DNA双链断裂的情况下，实现特定碱基的转换	编辑范围有限；在编辑窗口中发生旁观者编辑