Exploring the Trans‐Cleavage Activity of CRISPR‐Cas12a (cpf1) for the Development of a Universal Electrochemical Biosensor

Abstract

An accurate, rapid, and cost‐effective biosensor for the quantification of disease biomarkers is vital for the development of early‐diagnostic point‐of‐care systems. The recent discovery of the trans‐cleavage property of CRISPR type V effectors makes CRISPR a potential high‐accuracy bio‐recognition tool. Herein, a CRISPR‐Cas12a (cpf1) based electrochemical biosensor (E‐CRISPR) is reported, which is more cost‐effective and portable than optical‐transduction‐based biosensors. Through optimizing the in vitro trans‐cleavage activity of Cas12a, E‐CRIPSR was used to detect viral nucleic acids, including human papillomavirus 16 (HPV‐16) and parvovirus B19 (PB‐19), with a picomolar sensitivity. An aptamer‐based E‐CRISPR cascade was further designed for the detection of transforming growth factor β1 (TGF‐β1) protein in clinical samples. As demonstrated, E‐CRISPR could enable the development of portable, accurate, and cost‐effective point‐of‐care diagnostic systems.

Integrating CRISPR-Cas12a with a DNA circuit as a generic sensing platform for amplified detection of microRNA

“…Therefore, CRISPR-CHA was able to decrease the LOD by approximately 6 orders of magnitude compared with the classical CHA assay. Moreover, the LOD of CRISPR-CHA is 3–4 orders of magnitude lower than that of Cas13a-based miRNA assays, 18 , 19 providing an ultrasensitive method for the analysis of miRNA biomarkers.…”

“…The versatility of CRISPR-CHA has been demonstrated in the isothermal detection of miRNA biomarkers, including miR-21, miR-141 and miR-155 with sub-femtomolar sensitivity, which is significantly better than that of Cas13a-based methods. 18 , 19 As the gold standard for miRNA assay, the quantitative polymerase chain reaction requires multiple enzymes, complicated primer design and thermal cycles. 42 In contrast, CRISPR-CHA achieves the isothermal detection of miRNA with only one enzyme.…”

An Integrated Multi‐Function Heterogeneous Biochemical Circuit for High‐Resolution Electrochemistry‐Based Genetic Analysis

“…Thec oncatemer is the amplified product serving as the signaling strand for signal transduction based on the multiple Ss trands.F inally,t o acquire the molecular information, am icro-fabricated electrochemical single-use sensing array ( Figure S4) is applied to transduce the molecular signal into physicochemical signal through electrochemistry ( Figure 1d). [13] In order to probe the molecular signal through the output elongated concatemer, asurface capture strand modified with athiol group (SH-P1'), tethered on the gold working electrode through Au-S bond, is complementary to the primer sequence.T herefore,a ny primer,e ither going through the biochemical circuit or not, can be captured onto the sensor.Asignaling probe (S'-MB), complementary to the signaling strand (S) and containing am ethylene blue electrochemical tag, is further introduced into the system. Only with the presence of the signaling concatemer,m ultiple copies of signaling probes can be hybridized onto one concatemer generated by one copy of target originally,amplifying the electrochemical signal.…”

CRISPR Mediated Biosensing Toward Understanding Cellular Biology and Point‐of‐Care Diagnosis

“…This detection strategy, combined with ar olling polymerase amplification, was applied to the detection of human papillomavirus with attomolar sensitivity.O ther than using af luorescent transduction signal to probe the ssDNAnase activity of Cas12a, ap ortable and cost-effective transducing strategy based on electrochemistry has also been demonstrated (Figure 2b). [34] Am ethylene blue linked ssDNAwas tethered onto the gold working electrode surface of ad isposable screen-printed Angewandte Chemie Minireviews 20760 www.angewandte.org sensor.Through probing the electrochemical signal change of methylene blue,t he trans-cleavage activity of CRISPR Cas12a was indicated as ac urrent output. [35] Thed eveloped biosensor presented abroad dynamic range crossing pm to mm level for the detection of viral nucleic acids in buffer solutions and human serum samples.Asimilar biosensing strategy was also applied to the development of assaying Ebola RNAvirus through aC RISPR-Cas13a based integrated microfluidic based fluorescent detection system.…”

“…[38] Upon the programmable recognition of target miRNAt hrough Ca-s13a-crRNA, biotin and 6-fluorescein amidite (6-FAM) Adapted with permissionf rom ref. [34].C opyright 2019 John Wiley and Sons. (c) Cas13a-crRNA is programmedt orecognize miRNA in amicrofluidic electrochemical system.A nRNA reporter linked with 6-FAM and biotin is applied to probe the trans-cleavage activity of Cas13a.…”