Detection Dynamic and Development Tendency of Agricultural Gene Editing Products

doi:10.19586/j.2095-2341.2024.0112

Current Biotechnology ›› 2024, Vol. 14 ›› Issue (5): 712-723.DOI: 10.19586/j.2095-2341.2024.0112

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Detection Dynamic and Development Tendency of Agricultural Gene Editing Products

Jing WANG(), Haitao GUAN, Xiaolei ZHANG, Baohuai WANG, Baohai LIU(), Hongtao WEN()

Quality and Safety Institute of Agricultural Products，Heilongjiang Academy of Agricultural Sciences，Quality Testing Center of Cereals and Their Products （Harbin），Ministry of Agriculture and Rural Affairs，Key Laboratory of Quality and Safety of Cereals and Their Products，State Administration for Market Regulation，Harbin 150086，China

Received:2024-06-11 Accepted:2024-08-01 Online:2024-09-25 Published:2024-10-22
Contact: Baohai LIU,Hongtao WEN

农业基因编辑产品检测动态及发展趋势

王晶(), 关海涛, 张晓磊, 王堡槐, 刘宝海(), 温洪涛()

黑龙江省农业科学院农产品质量安全研究所，农业农村部谷物及制品质量检验测试中心（哈尔滨），国家市场监督管理总局重点实验室（谷物及制品质量与安全），哈尔滨 150086

通讯作者: 刘宝海,温洪涛
作者简介:王晶E-mail: buyijingjing@163.com；
基金资助:
科技创新2030-重大项目子课题(2022ZD040190803);黑龙江省农业科技创新跨越工程农业科技基础创新优青项目(CX22YQ08);黑龙江省自然科学基金优秀青年项目(YQ2022C032);黑龙江省省属科研院所科研业务费青年项目(CZKYF2024-1-C001);国家市场监督管理总局重点实验室（谷物及制品质量与安全）开放课题资助项目

Abstract

Abstract:

Gene editing technology is one of the essential biological breeding technologies. With the rapid development of biological breeding industrialization， agricultural gene editing products have shown a rapid growth trend. However， the speed of development of its identification and traceability detection technology cannot correspond to intellectual property protection and regulatory requirements. This seriously restricts the development of the industry. In this paper， we focused on the main gene editing systems， the technology advantages of various gene editing systems， and target editing types. Moreover， we analyzed the characteristics， advantages， and disadvantages of detection technology for gene editing products. Furthermore， three suggestions， innovation of detection technology， integration of detection and evaluation technology， and supervision technology system refinement， were proposed to provide reference for the research on the identification， traceability technology of agricultural gene editing products and technical support for improving the supervision system of agricultural biotechnology products.

Key words: gene editing, CRISPR/Cas technology, biological breeding, gene editing detection technology

摘要：

基因编辑技术是重要的生物育种技术之一。随着生物育种产业化的高速发展，农业基因编辑产品呈现快速增长态势，然而其标识溯源检测技术的研发速度难以匹配知识产权保护和安全监管的需求，严重制约了产业的发展，亟需研发与现行检测体系相适应的基因编辑产品检测技术与方法。从主要的基因编辑系统和技术优势、靶点编辑类型着手，分析了不同类型基因编辑产品检测技术的特征和优缺点，提出了检测技术创新、检测与评价技术融合、监管技术体系细化三方面建议，以期为农业基因编辑产品标识、溯源技术研究提供参考，为完善我国农业生物技术产品监管体系提供技术支撑。

关键词: 基因编辑, CRISPR/Cas技术, 生物育种, 基因编辑产品检测技术

CLC Number:

Jing WANG, Haitao GUAN, Xiaolei ZHANG, Baohuai WANG, Baohai LIU, Hongtao WEN. Detection Dynamic and Development Tendency of Agricultural Gene Editing Products[J]. Current Biotechnology, 2024, 14(5): 712-723.

王晶, 关海涛, 张晓磊, 王堡槐, 刘宝海, 温洪涛. 农业基因编辑产品检测动态及发展趋势[J]. 生物技术进展, 2024, 14(5): 712-723.

Figures/Tables 2

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分类	方法	技术特征	优点	缺点	灵敏度/识别碱基	参考文献
基于普通PCR方法	PCR/限制性核酸内切酶实验	编辑位点位于限制性酶切位点	简单、快速、经济，可检测纯合和杂合突变体	受限于原始序列	-	[52]
	CAPS衍生的方法和indCAPS方法	编辑位点可以利用错配引物创制限制性酶切位点，利用网络工具indCAPS设计含indel的dCAPS引物	对于不同类型的indel具有更好的灵活性	需要设计特别的引物区分一致的indel等位	-	[53]
	T7核酸内切酶Ⅰ实验	T7核酸内切酶降解野生型和突变型形成的错配的杂合双链	简单、快速、经济，能够检测杂合突变	灵敏度低，不能检测纯合突变	-	[52, 54]
	连接检测反应	使用高保真DNA连接酶将完全匹配的碱基对探针连接在两个合成寡核苷酸的缺口连接处时，等位基因特异性连接产物可以通过使用不同长度的引物和/或荧光标签修饰来唯一区分	低成本、精准、可靠，可以区分纯合与杂合基因型和多个等位基因	不能用于突变筛选	1 bp	[55]
	混合异源双链迁移率实验	基于有错配和无错配的DNA分子的差异迁移率，提供了一种简单方便的方法在测序确认之前辨别杂合生物体	可快速、有效地、筛选相关突变，简化纯合突变体分离鉴定过程	仅用于纯合突变体筛选	-	[56]
	单链构象多态性	检测单链核酸，PCR扩增产物变性形成单链，非变性PAGE胶检测	可鉴定纯合突变	片段小于300 bp，不能区分编辑类型	10%，2 bp	[57]
	基于聚丙烯酰胺凝胶电泳的方法	同源双链DNA在天然PAGE中的迁移速度快于异源双链DNA	简单、快速、经济，能够检测纯合和杂合突变	耗时长、低通量	0.5%，2 bp	[58]
	限制性片段长度多态性结合一代测序技术	取决于靶基因内是否产生限制性内切酶的识别位点	可测定突变类型	更费时、费力	-	[59]
	扩增片段长度多态性结合一代测序技术	利用限制性内切酶水解基因组DNA，再使双链人工接头的酶切片段相连接，作为扩增反应的模板DNA，接头的互补链为引物进行预扩增，通过聚丙烯酰胺凝胶电泳分离检测	分辨率高、稳定性好、效率高	费用高，对DNA的纯度和内切酶的质量要求很高	-	[60]
	临界温度退火PCR	PCR中临界退火温度抑制引物与模板的不匹配退火，抑制扩增子产生	简单、快速、经济，能够检测纯合突变	需要设计特异引物耗时费力	20%，1 bp	[61]
	特异性末端介导的聚合酶链式反应	设计产生带有特定末端部分的分子构建体，根据是否能够自折叠形成引物启动PCR扩增定性定量鉴定	能够定性定量检测单碱基突变，成本低、快速	引物设计复杂	30拷贝·反应^-1	[62]
基于qPCR方法	实时荧光PCR和标记的实验	针对突变位点设计不同修饰类型探针，结合qPCR技术	简单、有效、灵敏	不能明确突变中核酸的变化	<10%，1 bp	[63-64]
	高分辨率熔解曲线分析技术	纯合DNA具有独特的退火温度，而突变的杂合DNA具有较低的熔化温度	快速有效检测SNP和indel类型的编辑	需要特殊的仪器，灵敏度受扩增产物长度影响	4.7%，2 bp	[65]
	基于聚合酶链式反应的高分辨片段分析方法	通过设计大小不同并用不同荧光团标记的PCR扩增子来对具有一个以上靶向基因的样品进行多重分析	灵敏度高，可检测到小至1 bp，能够区分多个突变的等位基因	大小相同的indel等位不能共同被洗脱和分辨	2%，1 bp	[66]
	简单等位基因辨别聚合酶链式反应	利用一种位点特异性反向引物，通用荧光探针和猝灭剂，以及热启动DNA聚合酶	快速、精准、低成本	需要提前优化PCR反应条件	0.4 ng·10 μL^-1	[67]
基于数字PCR方法	数字PCR方法	将样品分割到许多单独的qPCR反应中，阴性结果用于生成样品中目标分子精确数量的绝对结果，无须参考标准品或内源性对照品	精准、低含量核酸加工食品、多倍体	成本高，需要特殊设备，不适用于大片段突变	1/100 000	[64]
基于测序技术	全基因组测序	建库进行高通量测序	鉴定突变打靶、打靶类型和脱靶	成本高、耗时，可能存在内生菌或病原物序列信息的污染等	-	[68]
基于测序技术	Sanger测序技术	第一代测序技术	准确鉴定突变位点	成本高、耗时、费力	-	[66]
基于其他技术	CRISPR/Cas系统	利用CRISPR/Cas12特异性识别编辑位点和非特异性切割的功能	低成本、条件温和、操作简单、灵敏度高、能实现快速检测	可能存在脱靶效应导致假阳性或假阴性，需要PAM/PFS识别序列，难以标准化，需要扩增过程	12 拷贝·μL^-1，1 bp	[69]

分类	方法	技术特征	优点	缺点	灵敏度/识别碱基	参考文献
基于普通PCR方法	PCR/限制性核酸内切酶实验	编辑位点位于限制性酶切位点	简单、快速、经济，可检测纯合和杂合突变体	受限于原始序列	-	[52]
	CAPS衍生的方法和indCAPS方法	编辑位点可以利用错配引物创制限制性酶切位点，利用网络工具indCAPS设计含indel的dCAPS引物	对于不同类型的indel具有更好的灵活性	需要设计特别的引物区分一致的indel等位	-	[53]
	T7核酸内切酶Ⅰ实验	T7核酸内切酶降解野生型和突变型形成的错配的杂合双链	简单、快速、经济，能够检测杂合突变	灵敏度低，不能检测纯合突变	-	[52, 54]
	连接检测反应	使用高保真DNA连接酶将完全匹配的碱基对探针连接在两个合成寡核苷酸的缺口连接处时，等位基因特异性连接产物可以通过使用不同长度的引物和/或荧光标签修饰来唯一区分	低成本、精准、可靠，可以区分纯合与杂合基因型和多个等位基因	不能用于突变筛选	1 bp	[55]
	混合异源双链迁移率实验	基于有错配和无错配的DNA分子的差异迁移率，提供了一种简单方便的方法在测序确认之前辨别杂合生物体	可快速、有效地、筛选相关突变，简化纯合突变体分离鉴定过程	仅用于纯合突变体筛选	-	[56]
	单链构象多态性	检测单链核酸，PCR扩增产物变性形成单链，非变性PAGE胶检测	可鉴定纯合突变	片段小于300 bp，不能区分编辑类型	10%，2 bp	[57]
	基于聚丙烯酰胺凝胶电泳的方法	同源双链DNA在天然PAGE中的迁移速度快于异源双链DNA	简单、快速、经济，能够检测纯合和杂合突变	耗时长、低通量	0.5%，2 bp	[58]
	限制性片段长度多态性结合一代测序技术	取决于靶基因内是否产生限制性内切酶的识别位点	可测定突变类型	更费时、费力	-	[59]
	扩增片段长度多态性结合一代测序技术	利用限制性内切酶水解基因组DNA，再使双链人工接头的酶切片段相连接，作为扩增反应的模板DNA，接头的互补链为引物进行预扩增，通过聚丙烯酰胺凝胶电泳分离检测	分辨率高、稳定性好、效率高	费用高，对DNA的纯度和内切酶的质量要求很高	-	[60]
	临界温度退火PCR	PCR中临界退火温度抑制引物与模板的不匹配退火，抑制扩增子产生	简单、快速、经济，能够检测纯合突变	需要设计特异引物耗时费力	20%，1 bp	[61]
	特异性末端介导的聚合酶链式反应	设计产生带有特定末端部分的分子构建体，根据是否能够自折叠形成引物启动PCR扩增定性定量鉴定	能够定性定量检测单碱基突变，成本低、快速	引物设计复杂	30拷贝·反应^-1	[62]
基于qPCR方法	实时荧光PCR和标记的实验	针对突变位点设计不同修饰类型探针，结合qPCR技术	简单、有效、灵敏	不能明确突变中核酸的变化	<10%，1 bp	[63-64]
	高分辨率熔解曲线分析技术	纯合DNA具有独特的退火温度，而突变的杂合DNA具有较低的熔化温度	快速有效检测SNP和indel类型的编辑	需要特殊的仪器，灵敏度受扩增产物长度影响	4.7%，2 bp	[65]
	基于聚合酶链式反应的高分辨片段分析方法	通过设计大小不同并用不同荧光团标记的PCR扩增子来对具有一个以上靶向基因的样品进行多重分析	灵敏度高，可检测到小至1 bp，能够区分多个突变的等位基因	大小相同的indel等位不能共同被洗脱和分辨	2%，1 bp	[66]
	简单等位基因辨别聚合酶链式反应	利用一种位点特异性反向引物，通用荧光探针和猝灭剂，以及热启动DNA聚合酶	快速、精准、低成本	需要提前优化PCR反应条件	0.4 ng·10 μL^-1	[67]
基于数字PCR方法	数字PCR方法	将样品分割到许多单独的qPCR反应中，阴性结果用于生成样品中目标分子精确数量的绝对结果，无须参考标准品或内源性对照品	精准、低含量核酸加工食品、多倍体	成本高，需要特殊设备，不适用于大片段突变	1/100 000	[64]
基于测序技术	全基因组测序	建库进行高通量测序	鉴定突变打靶、打靶类型和脱靶	成本高、耗时，可能存在内生菌或病原物序列信息的污染等	-	[68]
基于测序技术	Sanger测序技术	第一代测序技术	准确鉴定突变位点	成本高、耗时、费力	-	[66]
基于其他技术	CRISPR/Cas系统	利用CRISPR/Cas12特异性识别编辑位点和非特异性切割的功能	低成本、条件温和、操作简单、灵敏度高、能实现快速检测	可能存在脱靶效应导致假阳性或假阴性，需要PAM/PFS识别序列，难以标准化，需要扩增过程	12 拷贝·μL^-1，1 bp	[69]

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Detection Dynamic and Development Tendency of Agricultural Gene Editing Products

农业基因编辑产品检测动态及发展趋势

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