Enhancement and inactivation effect of CRISPR/Cas12a via extending hairpin activators for detection of transcription factors.
Yu JiangXinmei QianMingyu ZhengKeqing DengChunxiang LiPublished in: Mikrochimica acta (2023)
An enhancement effect for the activation of CRISPR/Cas12a (CRISPR = clustered regularly interspaced short palindromic repeats; Cas = CRISPR-associated) was discovered. That was, a hairpin model with dangling 5' end complementary to crRNA (CRISPR RNA) greatly improved the activity of CRISPR/Cas12a after extention of two random sequences. But, the corresponding intact hairpin without PAM (protospacer adjacent motif) or suboptimal PAM sequences was completely inactive to CRISPR/Cas12a because of the superhigh stability of intact hairpin. According to the finding, a CRISPR/Cas12a-based strategy coupled with a signal reported system was designed for transcription factors detection. By using mono-labeled ssDNA (single-stranded DNA) as reporter and two newly synthesized N-C (nitrogen-doped carbon) nanosheets as scavenger to eliminate the fluorescent background, the strategy realized the detection of NF-ĸB p50 (p50 subunit of nuclear factor kappa-B) with a linear detection range of 0.8 - 2000.0 pM and a LOD of 0.5 pM. The discovery of "enhancement and inactivation effect" not only deepened insight into CRISPR/Cas12a but also broadened the practical application of CRISPR/Cas systems for the molecular detection and disease diagnostics.
Keyphrases
- crispr cas
- genome editing
- nuclear factor
- transcription factor
- label free
- loop mediated isothermal amplification
- toll like receptor
- real time pcr
- air pollution
- particulate matter
- heavy metals
- risk assessment
- inflammatory response
- computed tomography
- single molecule
- quantum dots
- high throughput
- pet imaging
- positron emission tomography
- gold nanoparticles
- living cells
- cell proliferation