CRISPR/Cas12a-based biosensing platform for precise and efficient screening of CRISPR/Cas9-induced biallelic mutants

Guohui Xiao; Shuyan Liu; Houming Liu; Xing He; Su Zhang; Zhihang Liang; Huixin Guo; Min Ou; Lin Zhou; Lei Liu; Tianyu Zhang; Guoliang Zhang

doi:10.1016/j.talanta.2019.120613

CRISPR/Cas12a-based biosensing platform for precise and efficient screening of CRISPR/Cas9-induced biallelic mutants

Talanta. 2020 Apr 1:210:120613. doi: 10.1016/j.talanta.2019.120613. Epub 2019 Dec 7.

Authors

Guohui Xiao¹, Shuyan Liu², Houming Liu², Xing He², Su Zhang², Zhihang Liang³, Huixin Guo⁴, Min Ou², Lin Zhou⁴, Lei Liu², Tianyu Zhang⁵, Guoliang Zhang⁶

Affiliations

¹ Guangdong Key Laboratory of Emerging Infectious Diseases, Shenzhen Third People's Hospital, National Clinical Research Center for Tuberculosis, Southern University of Science and Technology, Shenzhen, 518112, China; State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.
² Guangdong Key Laboratory of Emerging Infectious Diseases, Shenzhen Third People's Hospital, National Clinical Research Center for Tuberculosis, Southern University of Science and Technology, Shenzhen, 518112, China.
³ Clinical Laboratory Diagnostics, Guangdong Medical University, Dongguan, 524023, China.
⁴ Guangdong Key Laboratory of Emerging Infectious Diseases, Shenzhen Third People's Hospital, National Clinical Research Center for Tuberculosis, Southern University of Science and Technology, Shenzhen, 518112, China; Guangdong Centre for Tuberculosis Control, Guangzhou, 510430, China.
⁵ State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.
⁶ Guangdong Key Laboratory of Emerging Infectious Diseases, Shenzhen Third People's Hospital, National Clinical Research Center for Tuberculosis, Southern University of Science and Technology, Shenzhen, 518112, China. Electronic address: [email protected].

PMID: 31987174
DOI: 10.1016/j.talanta.2019.120613

Abstract

CRISPR/Cas9 is a robust tool to manipulate genes in a wide range of species. Although several methods are introduced to identify the CRISPR/Cas9-induced mutations, they are labor-intensive, costly, and not easy to use or were sequence-limited. Moreover, few of them could identify the biallelic mutants that are the desired outcomes of targeted mutagenesis. Recently, a CRISPR/Cas12a-mediated biosensing platform was developed to detect nucleic acids based on the collateral DNA cleavage activity of Cas12a; it was highly sensitive, specific, rapid, and cost-efficient for genotyping, mutation detection, and single nucleotide polymorphism (SNP) identification, thereby deeming it as an innovative method for screening the CRISPR/Cas9-induced biallelic mutants. Thus, the CRISPR/Cas12a-based biosensing platform has been successfully utilized for screening 23 CRISPR/Cas9-induced biallelic mutants in Thp-1 cells, which were also confirmed by direct sequencing and ELISA. The precision and efficiency of CRISPR/Cas12a-based biosensing platform make it a promising tool for screening of CRISPR/Cas9-induced biallelic mutants in the future.

Keywords: Biallelic mutants; CRISPR/Cas12a; CRISPR/Cas9; Mutants screening.

MeSH terms

Biosensing Techniques*
CRISPR-Cas Systems / genetics*
Humans
Mutation
RNA / genetics
THP-1 Cells

Substances

RNA