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

doi:10.19586/j.2095-2341.2026.0028

Current Biotechnology ›› 2026, Vol. 16 ›› Issue (2): 356-364.DOI: 10.19586/j.2095-2341.2026.0028

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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

Abstract

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

CLC Number:

Q819

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.

Figures/Tables 7

References 21

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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

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

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

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