A CRISPR/Cas12a-derived Biosensing Platform For The Detection Of Diverse Small Molecules With The Assistance Of Allosteric Transcription Factor

It is of great significance to detect small molecules in environmental monitoring,food safety and medical diagnosis.The routine detection of small molecules mainly relies on spectra and separation equipment,but these technologies limit the application due to the high cost of equipment and cumbersome operation.Allosteric transcription factor(aTF)is a kind of regulatory protein widely distributed in bacteria,which usually contains two domains:effector binding domain(EBD)and DNA binding domain(DBD).Target molecule usually induces a conformational change of aTF by the direct binding to aTF effector binding domain,then enhances or attenuates the double-stranded DNA(dsDNA)-binding capacity of aTF and regulates the transcription and expression of target gene.Therefore,the small molecule signal can be converted into readable dsDNA signal by aTF.It is a very valuable recognition element in small molecule detection.CRISPR/Cas system is a component of acquired immune system ubiquitous in most bacteria and archaea.Since 2003,CIRPSR/Cas9 has been developed as an efficient gene editing tool and was widely used in life science research.In 2018,the trans-cleavage activity of CRISPR/Cas 12a was reported.Under the guide of crRNA,dsDNA can unleash the trans-cleavage activity of Cas 12a to efficiently cleavage any sequence of single-stranded DNA(ssDNA).Based on the trans-cleavage activity of CRISPR/Cas 12a,this study established a small molecule detection system named CaT-Smelor by using the fluorescence reporting system and immobilized aTF.In this study,p-hydroxybenzoic acid that is used as a food preservatives and uric acid which is a diagnostic marker for gout were tested by using this detection system.Our results indicate that CaT-Smelor can specifically distinguish p-hydroxybenzoic acid and uric acid from their corresponding structural analogues with high concentration.The target small molecule can be detected qualitatively by fluorescence signal.Meanwhile,CaT-Smelor performed quantitative detection of p-hydroxybenzoic acid and uric acid at the nanomolar level.The linear relationship between fluorescence intensity and uric acid concentration is y=10.7x+1289.8(R2= 0.992),and the linear curve between fluorescence intensity and hydroxybenzoic acid concentration is y=6.3x+656.6(R2 =0.999).Moreover,20 clinical serum samples were analyzed by CaT-Smelor,HPLC and medical biochemical analyzer,and the detection results were compared.It was found that the results of these three methods were nearly identical.But CaT-Smelor has obvious advantages in both cost and time compared with HPLC and medical biochemical analyzer.Finally,this small molecule detection method based on CRISPR/Cas12a and aTF was established.Simple,sensitive,rapid and low-cost detection of target small molecules was achieved.Moreover,CRISPR systerm was extended to the field of small molecule detection.Because the detection system is very small,high throughput detection can be achieved in 96-well plate or 384-well plate.In addition to clinical serum detection,this method can also be used for the detection of contaminants in food and soil.