Ultrasensitive detection of gene-PIK3CAH1047R mutation based on cascaded strand displacement amplification and trans-cleavage ability of CRISPR/Cas12a

Yuanyi Deng; Gaihua Cao; Xiaolong Chen; Mei Yang; Danqun Huo; Changjun Hou

doi:10.1016/j.talanta.2021.122415

Ultrasensitive detection of gene-PIK3CA^H1047R mutation based on cascaded strand displacement amplification and trans-cleavage ability of CRISPR/Cas12a

Talanta. 2021 Sep 1:232:122415. doi: 10.1016/j.talanta.2021.122415. Epub 2021 Apr 20.

Authors

Yuanyi Deng¹, Gaihua Cao¹, Xiaolong Chen¹, Mei Yang¹, Danqun Huo², Changjun Hou³

Affiliations

¹ Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044, PR China.
² Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044, PR China; Chongqing Key Laboratory of Bio-perception & Intelligent Information Processing, School of Microelectronics and Communication Engineering, Chongqing University, Chongqing, 400044, PR China. Electronic address: [email protected].
³ Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044, PR China; Chongqing Key Laboratory of Bio-perception & Intelligent Information Processing, School of Microelectronics and Communication Engineering, Chongqing University, Chongqing, 400044, PR China. Electronic address: [email protected].

PMID: 34074403
DOI: 10.1016/j.talanta.2021.122415

Abstract

Low abundance gene-PIK3CA^H1047R mutation detection is crucial for the clinical diagnosis and treatment of breast cancer. Here, a fluorescent biosensor which combines cascaded strand displacement amplification (C-SDA) and trans-cleavage ability of CRISPR/Cas12a was established to ultra-sensitively detect gene-PIK3CA^H1047R mutation. The mutated gene-PIK3CA^H1047R can combine with complementary sequence to form an intact recognition site for endonuclease FspI. Mediated by FspI, it breaks at the mutation site to produce DNA fragment to trigger SDA or C-SDA. Then, the fluorescent biosensors based on SDA-CRISPR/Cas12a or C-SDA-CRISPR/Cas12a were constructed. Compared with biosensor based on SDA-CRISPR/Cas12a (5 pM), the minimum detection of the biosensor based on C-SDA-CRISPR/Cas12a is reduced two orders of magnitude (50 fM). In range of 0.001%-50%, we achieved the ultrasensitive detection of gene-PIK3CA^H1047R mutation low to 0.001%. Besides, the proposed biosensor works well in human serum samples, showing its application potential in low-abundance gene-PIK3CA^H1047R mutation detection.

Keywords: CRISPR/Cas12a; Fluorescent biosensor; Gene-PIK3CA(H1047R) mutation; Strand displacement amplification (SDA).

MeSH terms

Biosensing Techniques*
CRISPR-Cas Systems / genetics
Class I Phosphatidylinositol 3-Kinases / genetics
Clustered Regularly Interspaced Short Palindromic Repeats*
Humans
Mutation

Substances

Class I Phosphatidylinositol 3-Kinases
PIK3CA protein, human