GeoCas9介导的无sgRNA的短双链DNA快速单链化方法及其性能表征

doi:10.19586/j.2095-2341.2026.0028

生物技术进展 ›› 2026, Vol. 16 ›› Issue (2): 356-364.DOI: 10.19586/j.2095-2341.2026.0028

• 研究论文 • 上一篇

GeoCas9介导的无sgRNA的短双链DNA快速单链化方法及其性能表征

李欣然¹(), 王晨怡¹, 齐鑫¹, 郭亚宁², 李付凯¹, 周剑¹, 杨梦瑞¹, 李亮¹, 李凯¹()

^1.中国农业科学院农业质量标准与检测技术研究所，北京 100081
^2.天津农学院食品科学与生物工程学院，天津 300384

收稿日期:2026-01-16 接受日期:2026-01-27 出版日期:2026-03-25 发布日期:2026-04-27
通讯作者: 李凯
作者简介:李欣然 E-mail: lllxinran_05@163.com；
基金资助:
国家自然科学基金项目(32402239)

GeoCas9-mediated sgRNA-free Short Double-stranded DNA Rapid Single-stranded Method and its Performance Characterization

Xinran LI¹(), Chenyi WANG¹, Xin QI¹, Yaning GUO², Fukai LI¹, Jian ZHOU¹, Mengrui YANG¹, Liang LI¹, Kai LI¹()

^1.Institute of Quality Standard and Testing Technology for Agro-Products of Chinese Academy of Agricultural Sciences，Beijing 100081，China
^2.College of Food Science and Bioengineering，Tianjin Agricultural University，Tianjin 300384，China

Received:2026-01-16 Accepted:2026-01-27 Online:2026-03-25 Published:2026-04-27
Contact: Kai LI

摘要/Abstract

摘要：

短双链DNA（short double-stranded DNA, dsDNA）的高效单链化是分子诊断、基因编辑及DNA纳米结构构建等领域中关键的前处理步骤。然而，现有方法主要依赖高温变性或化学变性策略，存在操作复杂、可逆性差以及可能损伤DNA完整性等局限。报道了一种基于CRISPR相关蛋白GeoCas9的体外dsDNA快速单链化新策略。研究表明，在无sgRNA引导条件下，GeoCas9即可结合并解旋长度为19~55 bp的短dsDNA底物，在常温条件下实现非切割、可逆且高效的单链化过程。系统评估了蛋白浓度、蛋白与底物摩尔比、反应时间以及dsDNA长度对单链化效率的影响，并确定了最优反应参数为：GeoCas9蛋白终浓度50 nmol·L^-1，蛋白与dsDNA底物摩尔比为1∶1（dsDNA浓度50 nmol·L^-1）。在优化条件下，该方法可在温和环境中实现快速且稳定的双链解旋，同时保持DNA结构的完整性。研究拓展了Cas9蛋白的功能认知，为精密核酸操控与下游生物分析提供了一种简便、高效且具有良好可控性的技术路径。

关键词: GeoCas9蛋白, 短双链DNA, DNA单链化, 非变性解旋

Abstract:

Efficient conversion of short double?stranded DNA （dsDNA） into single?stranded DNA is a critical pretreatment step in molecular diagnostics， genome editing， and DNA nanostructure construction. However， existing methods mainly rely on thermal or chemical denaturation strategies， which suffer from limitations such as complex operation， poor reversibility， and potential damage to DNA integrity. Herein， we reported a novel rapid in vitro strategy for the single?stranding of short dsDNA based on the CRISPR?associated protein GeoCas9. Results showed that GeoCas9 can bind and unwind short dsDNA substrates with lengths ranging from 20 to 60 bp in the absence of sgRNA guidance， enabling a non?cleaving， reversible， and highly efficient single?stranding process under ambient conditions. We systematically evaluated the effects of protein concentration， protein?to?substrate molar ratio， reaction time， and dsDNA length on the single?stranding efficiency， and determined the optimal reaction parameters： a final GeoCas9 concentration of 50 nmol·L^-1 and a protein?to?dsDNA molar ratio of 1∶1 （dsDNA concentration 50 nmol·L^-1）. Under optimized conditions， this method achieves rapid and stable duplex unwinding in a mild environment while maintaining DNA structural integrity. This study expands the functional understanding of Cas9 proteins and provides a simple， efficient， and well?controllable technical route for precise nucleic acid manipulation and downstream bioanalysis.

Key words: GeoCas9 protein, short double-stranded DNA, DNA single stranding, non-denatured unwinding

中图分类号:

Q819

李欣然, 王晨怡, 齐鑫, 郭亚宁, 李付凯, 周剑, 杨梦瑞, 李亮, 李凯. GeoCas9介导的无sgRNA的短双链DNA快速单链化方法及其性能表征[J]. 生物技术进展, 2026, 16(2): 356-364.

Xinran LI, Chenyi WANG, Xin QI, Yaning GUO, Fukai LI, Jian ZHOU, Mengrui YANG, Liang LI, Kai LI. GeoCas9-mediated sgRNA-free Short Double-stranded DNA Rapid Single-stranded Method and its Performance Characterization[J]. Current Biotechnology, 2026, 16(2): 356-364.

图/表 7

表1 sgRNA和靶标DNA序列

Table 1 Sequences of sgRNA and target DNA

名称	序列（5'→3'）	序列长度/bp
sgRNA	5'-GUCACCUCCAAUGACUAGUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3'	—
FAM-dsDNA-19	5'-CTCCAATGACTAGTCAGGG-3'	19
BHQ1-dsDNA-19	5'-CCCTGACTAGTCATTGGAG-3'	19
FAM-dsDNA-20	5'-CTCCAATGACTAGTCAGGGC-3'	20
BHQ1-dsDNA-20	5'-GCCCTGACTAGTCATTGGAG-3'	20
FAM-dsDNA-27	5'-GTCACCTCCAATGACTAGTCAGGGCAA-3'	27
BHQ1-dsDNA-27	5'-TTGCCCTGACTAGTCATTGGAGGTGAC-3'	27
FAM-dsDNA-35	5'-AGTGCGTCACCTCCAATGACTAGTCAGGGCAATAG-3'	35
BHQ1-dsDNA-35	5'-CTATTGCCCTGACTAGTCATTGGAGGTGACGCACT-3'	35
FAM-dsDNA-45	5'-AGCAGCCAGTGCGTCACCTCCAATGACTAGTCAGGGCAATAGCGA-3'	45
BHQ1-dsDNA-45	5'-TCGCTATTGCCCTGACTAGTCATTGGAGGTGACGCACTGGCTGCT-3'	45
FAM-dsDNA-55	5'-AATGGAGCAGCCAGTGCGTCACCTCCAATGACTAGTCAGGGCAATAGCGAACCAC-3'	55
BHQ1-dsDNA-55	5'-GTGGTTCGCTATTGCCCTGACTAGTCATTGGAGGTGACGCACTGGCTGCTCCATT-3'	55

图1 FAM/BHQ1标记的DNA退火条件优化

Fig. 1 Optimization of annealing conditions for FAM/BHQ1-labeled DNA

图2 GeoCas9蛋白介导短双链DNA单链化验证A:完整反应体系；B:缺少sgRNA的反应体系；C:缺少GeoCas9蛋白的反应体系；D:缺少dsDNA-F/Q的反应体系；E:缺少GeoCas9/sgRNA反应体系；F:空白对照组

Fig. 2 GeoCas9 protein-mediated short dsDNA single-strand verification

图3 各组分对即时GeoCas9单链化效率的影响注：****表示各处理间在P<0.000 1水平有统计学差异；ns表示各处理间无统计学差异（P>0.05）。

Fig. 3 Influence of component on the single chain efficiency of instantaneous GeoCas9

图4 GeoCas9介导的短双链DNA单链化作用特异性分析

Fig. 4 Specificity analysis of GeoCas9-mediated short dsDNA single-stranded effect

图5 GeoCas9介导短双链DNA单链化效率的反应参数优化A:单链化效率与GeoCas9蛋白浓度优化；B:单链化效率与GeoCas9蛋白与短双链DNA摩尔比优化。*、**、***分别表示差异在P<0.05、P<0.01和P<0.001水平有统计学意义；ns表示差异无统计学意义（P>0.05）。

Fig. 5 Optimization of reaction parameters for GeoCas9-mediated single-stranded efficiency of short dsDNA

图6 GeoCas9对不同长度短双链DNA的单链化适用性分析

Fig. 6 Applicability of GeoCas9-mediated single-stranding to short dsDNA substrates of different lengths

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GeoCas9介导的无sgRNA的短双链DNA快速单链化方法及其性能表征

GeoCas9-mediated sgRNA-free Short Double-stranded DNA Rapid Single-stranded Method and its Performance Characterization

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