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International Edition: DOI: 10.1002/anie.201910772

Biosensors German Edition: DOI: 10.1002/ange.201910772

Exploring the Trans-Cleavage Activity of CRISPR-Cas12a (cpf1) for

the Development of a Universal Electrochemical Biosensor
Yifan Dai,* Rodrigo A Somoza, Liu Wang, Jean F. Welter, Yan Li, Arnold I Caplan,* and
Chung Chiun Liu*

Abstract: An accurate, rapid, and cost-effective biosensor for racy of the CRISPR system in targeting nucleic acids, owing
the quantification of disease biomarkers is vital for the to the complementarity-dependent CRISPR cleavage
development of early-diagnostic point-of-care systems. The event.[2] Utilizing collateral cleavage of the nonspecific
recent discovery of the trans-cleavage property of CRISPR ssDNA reporter (termed trans-acting cleavage), initiated by
type V effectors makes CRISPR a potential high-accuracy bio- the recognition and cleavage of the target DNA (termed cis-
recognition tool. Herein, a CRISPR-Cas12a (cpf1) based acting cleavage) by Cas-crRNA, CRISPR type III, V, and VI
electrochemical biosensor (E-CRISPR) is reported, which is RNA-guided nucleases (Csm6, Cas12a, Cas13) have been
more cost-effective and portable than optical-transduction- used for the detection of nucleic acids (RNA/DNA) through
based biosensors. Through optimizing the in vitro trans- fluorescent transduction systems.[3] Herein, we extend the
cleavage activity of Cas12a, E-CRIPSR was used to detect application of the CRISPR-Cas12a (cpf1) system to the
viral nucleic acids, including human papillomavirus 16 (HPV- development of an electrochemical biosensing system be-
16) and parvovirus B19 (PB-19), with a picomolar sensitivity. cause of its relative cost-efficiency and the portable nature of
An aptamer-based E-CRISPR cascade was further designed the transduction system compared with the fluorescent trans-
for the detection of transforming growth factor b1 (TGF-b1) ducer. Moreover, for a comprehensive and robust point-of-
protein in clinical samples. As demonstrated, E-CRISPR could care system, a potential universal biosensing platform that can
enable the development of portable, accurate, and cost- detect different categories of analytes is of high importance
effective point-of-care diagnostic systems. for clinical applications. Therefore, building upon the accu-
rate CRISPR-based sensing system, we further design a Ca-
Introduction s12a-based biosensing cascade as a strategy for protein
detection. Our study demonstrates a CRISPR-Cas12a (cpf1)
An accurate, rapid, and cost-effective sensing strategy for based electrochemical biosensing platform (E-CRISPR) for
the quantification of disease biomarkers is vital for the the detection of the major categories of biomolecules,
development of early-diagnostic point-of-care systems, fur- providing a potential deployable point-of-care system for
ther leading to personalized medicine and benefiting overall the healthcare industry.
human health. Electrochemistry-based biosensing platforms E-CRISPR is a simple endonuclease activity transduction
have been widely developed, owing to its rapid signal readout, method for CRISPR type III, V, VI nuclease-based sensing
affordable transduction element, and simple sensing plat- systems and enables the detection of multiple analyte classes.
form.[1] One of the critical challenges for such a sensing A disposable, micro-fabricated gold-based three-electrode
system is its accuracy. Recent robust developments of sensor with gold as working and counter electrodes and Ag/
CRISPR (clustered regularly interspaced short palindromic AgCl as the reference electrode was applied for the develop-
repeats) based gene editing systems demonstrated the accu- ment of the electrochemical biosensor (Supporting Informa-
tion, Figure S1).[4] The Cas12a-crRNA duplex is designed to
specifically recognize and cleave a target nucleic acid strand
[*] Y. Dai, Prof. C. C. Liu
based on the protospacer adjacent motif (PAM) sequence of
Department of Chemical and Biomolecular Engineering,
Electronics Design Center, Case Western Reserve University the target and complementarity between the target and the
Cleveland, OH 44106 (USA) crRNA (Figure 1 A).[5] PAM recognition depends on the
E-mail: yxd176@case.edu specific 5’-TTTN nucleic acid sequence located in the
cxl9@case.edu opposite strand (blue strand) to the recognition strand
Dr. R. Somoza, Prof. J. F. Welter, Prof. A. I Caplan (orange strand). Only upon the recognition of the PAM
Department of Biology, Skeletal Research Center & sequence by the Cas protein, the Cas protein, acting as a DNA
Center for Multimodal Evaluation of Engineered Cartilage, helicase, unwinds the target DNA. After the separation of the
Case Western Reserve University, Cleveland, OH 44106 (USA)
target strands, the complementarity (between crRNA and
E-mail: aic@case.edu
target) dependent cleavage activity can further be activated.[6]
Dr. L. Wang, Prof. Y. Li
Department of Genetics and Genome Sciences, School of Medicine, To achieve the electrochemical transduction of the CRISPR
Case Western Reserve University, Cleveland, OH 44106 (USA) detection signal, the cis-cleavage-initiated trans-cleavage
Supporting information and the ORCID identification number(s) for effect of Cas12a on the nonspecific ssDNA is probed through
the author(s) of this article can be found under: an electrochemical method. A nonspecific ssDNA reporter is
https://doi.org/10.1002/anie.201910772. designed with a methylene blue (MB) electrochemical tag for

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Figure 1. Principle of E-CRISPR. A) Cas12a (cpf1) performs crRNA-guided trans-cleavage of nonspecific ssDNA, initiated by the cis-cleavage of
specific DNA. B) Nonspecific ssDNA reporter with methylene blue tag immobilized on the gold electrode. C) In the presence of the target,
Cas12a-crRNA would initiate the trans-cleavage of the nonspecific ssDNA reporter, resulting in a low electrochemical current of methylene blue.
D) In the absence of the target, Cas12a-crRNA would not initiate the trans-cleavage of the nonspecific ssDNA reporter, resulting a high
electrochemical current of methylene blue. E) A representation of electrochemical current outputs in the absence and presence of the target.

signal transduction and a thiol moiety to tether on the sensor HPV16 was identified based on the TTTN PAM sequence
surface in order to acquire the signal electrically (Fig- required by the Cas12a endonuclease.[9] The electrochemical
ure 1 B).[7] Consequently, the electron transfer process be- biosensing platform was initially developed based on the Cas12a
tween the gold electrode and the redox active species on the endonuclease from Acidaminococcus sp. (AsCas12a).[10] We
ssDNA can be electrochemically initiated and transduced. first investigated the on-chip collateral cleavage performance
With the presence of the target, the Cas12a trans-cleavage based on the AsCas12a-crRNA duplex targeting the HPV-16
activity is activated, cleaving the MB-ssDNA reporter off the sequence. After assembling the HPV-16 and the AsCas12a-
electrode surface, therefore decreasing the MB signal trans- crRNA, the triplex complex was directly incubated on the
duced (Figure 1 C). In the absence of the target, the Cas12a ssDNA -reporter-coated electrode. Square wave voltammetry
trans-cleavage activity is silenced, retaining the MB-ssDNA (SWV) was applied to evaluate the MB signal, which was
reporter on the surface (Figure 1 D). A representation of decreased only in the presence of the cognate target with the
electrochemical signal output based on the conditions with- corresponding AsCas12a-crRNA (Figure 2 A).
out/with target is shown in Figure 1 E. The design of the MB-
ssDNA-coated electrode is generally applicable for any
CRISPR type III, V, and VI systems as a simple and cost- Evaluation of the Optimized Condition for On-Chip Trans-
effective CRISPR activity transduction strategy. Based on the Cleavage Activity
concept of E-CRISPR, we developed this platform as
a universal biosensing strategy for the detection of nucleic For biosensing, the detection sensitivity is critical due to
acid and protein. the low abundance of clinically relevant biomarkers in human
fluids.[11] For the E-CRISPR detection platform, the trans-
cleavage activity is the key for signal transduction, and
Results and Discussion therefore is critical to the sensitivity performance. We first
compared the on-chip trans-cleavage activity of another type
Verification of E-CRISPR for Nucleic Acid Detection of Cas12a protein, Lachnospiraceae bacterium ND2006
Cas12a (LbCas12a),[12] with AsCas12a. LbCas12a demon-
To examine the feasibility of the E-CRISPR for nucleic strated a more apparent and stable trans-cleavage response
acid detection, a human papilloma virus (HPV) subtype, within 5 min compared with that of AsCas12a (Figure 2 B).
HPV-16, which is critical to carcinogenesis,[8] was selected as LbCas12a presented a more robust trans-cleavage activity
the target. A target sequence in the L1-encoding gene of within the testing period using the same experimental

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Figure 2. Optimization of on-chip trans-cleavage activity. A) Representation of square wave voltammetry (SWV) evaluation of E-CRISPR in
response to HPV-16. Red curve represents the background signal of 50 nm of Cas12a-crRNA duplex. Black curve represents the signal generated
by the target-induced trans-cleavage activity of 50 nm of Cas12a-crRNA. B) Evaluation of 50 nm of Cas12a orthologues from Lachnospiraceae and
Acidaminococcus sp. on its activity for on-chip trans-cleavage activity based on the change of current between background signal and target-
Background signalTarget signal
mediated signal. DI% ¼ Background signal (red line: LbCas12a; black line: AsCas12a). C) Evaluation of trans-cleavage activity using 50 nm of
LbCas12a-crRNA-target triplex. D) Evaluation of the effect of the concentration of divalent metal ions on the trans-cleavage activity of the RuvC
domain using 50 nm of LbCas12a-crRNA-target triplex. E,F,G) SWV graphs of different lengths of surface ssDNA reporters based on 30 nm of
LbCas12a-crRNA-target triplex. H) Comparison of signal change from different lengths of surface ssDNA reporters. SWV graphs in these figures
present the result of a single test. Error bars in figures present the standard error (SE) based on at least three individual tests using at least three
different sensors.

conditions, therefore LbCas12a was selected for further E- important factor that may affect the Cas12a cleavage activity
CRISPR development. We further evaluated the possible is the divalent cation Mg2+ concentration in the testing
factors that may affect the trans-cleavage activity for on-chip solution.[13] Cas12a RuvC domain is known to cleave ssDNA
electrochemical test using HPV-16 as the target. The optimal through the two-metal ion mechanism,[14] in which the Mg2+
trans-cleavage period was investigated. The DI% continu- ions induce conformational coordination of the RuvC domain
ously increased with increasing incubation time for the and the ssDNA by shifting the spatial distribution of ssDNA
collateral cleavage event (Figure 2 C). It is interesting to around the RuvC active cutting center. Therefore, we
notice that trans-cleavage does not occur simultaneously with evaluated the effect of concentration of Mg2+ ions in the
cis-cleavage. The cis-cleavage of target strand is typically in vitro cleavage solution on the activity of trans-cleavage
finished within 30 min;[3a] however, the trans-cleavage func- function. The trans-cleavage activity was only activated with
tion remained active even after 3 hours (Figure 2 C), indicat- the presence of the Mg2+ ions in the testing solution (Fig-
ing that the target recognition initiated cis-cleavage activity of ure 2 D). Increasing concentration of Mg2+ cations up to
the Cas12a system is the activator for the trans-cleavage 15 mm demonstrated an enhanced trans-cleavage activity.
functional domain of the Cas12a endonuclease. Hence, an optimized Mg2+ concentration of 15 mm was
We further investigated the chemical environment of the selected for the preparation of the Cas12a-crRNA duplex.
Cas12a to optimize the trans-cleavage performance. An

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In order to perform an efficient surface-chemistry-based Moreover, a multi-component monolayer system (for exam-
trans-cleavage, the accessibility of Cas12a endonuclease to ple, ternary self-assembled monolayers) might also increase
the nonspecific ssDNA is important. Thus, we evaluated the sensitivity through tuning the molecular packing on the
effect of ssDNA reporter density on the electrode surface on surface.[16]
the variation of electrochemical signal before and after the Another interesting finding regarding the cleavage acces-
trans-cleavage activity. An ideal surface condition can pro- sibility is that Cas12a-crRNA-based trans-cleavage activity is
vide an optimized electrostatic environment for charged also significantly concentration dependent, as is the case for
phosphate backbones and the hydroxyl groups of the the analogue Cas9.[17] Different concentrations of Cas12a-
passivation agents to ensure an upright ssDNA surface, crRNA in response to a same target concentration were
facilitating the cleavage activity. The surface density of evaluated (Supporting Information, Figure S5). A high con-
ssDNA reporter was manipulated by the concentration of centration level (greater than 100 nm) in a 30 mL sample
the ssDNA reporter incubation solution. As shown in Fig- solution significantly decreased the activity of the Cas12a
ure S2 A in the Supporting Information, a high surface density endonuclease towards a nonspecific ssDNA reporter as the
of ssDNA reporter significantly decreased the change of large size of Cas12a hinders its diffusion in the testing
signal, because this high surface density decreased the solution. Hence, a relative minor change in current outputs
accessibility of Cas endonuclease to the ssDNA reporter, was observed when using a high concentration of Cas12a-
producing a steric hindrance effect, which limited the trans- crRNA. An optimized concentration for Cas12a-crRNA
cleavage activity. An ideal density was prepared using 1 mm of duplex trans-cleavage operation was identified to be 30 nm
ssDNA reporter and identified as 5.2  1014 mol mm2 (Sup- in a 30 mL solution.
porting Information, Figure S2 B), which created sufficient
space for Cas12a to perform collateral cleavage on the
electrode surface, providing a sufficient electrochemical E-CRISPR on Nucleic Acid Detection
signal change and ensuring an excellent detection resolution.
Other than the surface density, the length of the immo- Based on the optimized trans-cleavage conditions, we
bilized ssDNA reporter was also evaluated. We hypothesized evaluated the E-CRISPR platform for the detection of HPV-
that ssDNA reporters with different lengths might lead to 16. A broad dynamic range (pm to mm) of more than three
different cleavage efficiency due to the exposed length orders of magnitude was achieved with an IC50 value of
difference. Different lengths of ssDNA probes at the same 0.78 nm based on the samples prepared in the buffer solution
concentration were evaluated using the same reaction con- (Figure 3 A). The dose-dependent response curve demon-
ditions of E-CRISPR as investigated previously. Moreover, strated an average standard error (SE) of 2.16 % (n = 3),
the effect of passivation agents with different carbon chain indicating a reliable reproducibility. An experimental limit of
lengths may influence the electrostatic interaction between detection (LOD) of 50 pm was obtained. It is worth mention-
the phosphate backbones of ssDNA probes, therefore this was ing that this LOD surpasses the previously published LOD for
also evaluated for optimal cleavage activity (Supporting non-enzymatically amplified nucleic-acid detection by over
Information, Figure S3). The selected ssDNA and passivation two orders of magnitude.[3b] Moreover, the detection perfor-
agent pairs were then compared through the effect of lengths mance in a complex matrix was also evaluated. An IC50 value
on trans-cleavage activity. However, different lengths of in pooled human serum was 0.68 nm, which is comparable
ssDNA reporter only produce a minute variation (less than with the IC50 value (0.78 nm) in buffer solution, indicating
5 %) in signal change (Figure 2 H). We observed that for a great potential of E-CRISPR in the direct analysis of
a short ssDNA reporter (10 nt), the electrochemical oxidation biological samples. We also tested the effect of target length
current over the background current was larger than that of on the trans-cleavage signal by increasing the length of the
a long ssDNA strand because of its short contact-mediated HPV-16 targeting sequence from 40 mer to 100 mer in the L1-
electron tunneling distance to the electrode, resulting faster enconding region.[9] Compared with the detection perfor-
charge transfer kinetics (Figure 2 E). Therefore, the short mance of the 40 mer DNA target, the 100 mer DNA target
probe possessed a large baseline current. As for long presented a similar IC50 value (0.62 nm, Supporting Informa-
reporters (20 nt and 30 nt), they gave a relative low back- tion, Figure S6), indicating that the length of the target would
ground current (Figure 2 F,G), but the DI% of these long not interfere with the in vitro trans-cleavage activity of
reporters were comparable to that of short reporter. 20 nt Cas12a.
ssDNA reporter was selected for further application because To evaluate the generality of the detection strategy, we
of its relative greater degree of signal change and smaller used the E-CRISPR system to detect ssDNA erthrovirus,
standard error (Figure 2 H). With the completion of the Parvovirus B19 (PB-19), which is known to cause erythema
surface packing optimization, we tested the storage stability infectiosum in children and pregnant women.[18] A dynamic
of the optimized ssDNA electrode by storing at 4 8C in detection range from pm to mm was achieved with an IC50
a humidified environment (Supporting Information, Fig- value of 0.60 nm (Supporting Information, Figure S7 A). The
ure S4). A stable SWV signal was retained for around 3 days, percentage of signal change was similar to that of detection
which is a sufficient turnaround time for a clinical point-of- performance by HPV-16, indicating the on-chip trans-cleav-
care routine. If a longer storage stability is needed, a multi- age activity would not be affected by different targets.
component monolayer system can be applied as demonstrat- We further investigated the accuracy of the E-CRISPR
ed previously to maintain a high sensitivity over months.[15] platform. A scrambled sequence and PB-19 were applied to

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The clear differentiable SWV signal between mismatches at

different positions also suggests that the trans-cleavage
activity of Cas12a might be utilized to identify the position
of the mismatched base pairs as a biosensing strategy. Overall,
E-CRISPR demonstrates a sensitive, generalized, and cost-
effective platform for nucleic acid analysis.

Aptamer-Based E-CRISPR Cascade for Protein Detection

We next explored whether the E-CRISPR could be

repurposed as a protein detection platform by utilizing the
nucleic acid detection capability of E-CRISPR. For protein
detection, an ssDNA aptamer was used as the recognition
element for a protein of interest. An aptamer-based E-
CRISPR cascade was designed for protein detection (Fig-
ure 4), which allows the direct analysis of complex sample
Figure 3. E-CRISPR analysis of HPV-16. A) Dose-response curve of the
without any time-consuming processing procedures. A fixed
detection of HPV-16 in different matrixes (green line: 10 mm Tris
buffer containing 50 mm NaCl and 15 mm MgCl2 ; purple line: 100 %
concentration of aptamer is firstly applied to treat the sample
human serum). B) Selectivity study through comparison of the signal directly (Figure 4 A). Cas12a-crRNA is designed to specifi-
changes based on non-target nucleic acids (500 nm) with that of 1 nm cally recognize the aptamer. The E-CRISPR is then applied
of HPV-16 (n = 3, *P < 0.01, target signal vs. non-target signal). to determine the remaining concentration of aptamer in the
C) Target strands with mismatches at different positions, including sample (Figure 4 B). In the presence of the protein target, less
PAM region and crRNA complement at different positions: 1, 6, 11, aptamer would be captured and transduced by E-CRISPR,
and 16. D) Evaluation of the influence of mismatches at different
leading to a high electrochemical signal of the MB from the
positions on the E-CRISPR signal. A target concentration of 1 nm was
applied for all the targets (wild type (WT) and mismatched targets). ssDNA reporter. In the absence of the protein target, the
electrochemical signal would be lower due to the activation of
trans-cleavage activity by ssDNA target recognition (Fig-
evaluate the selectivity for HPV-16 detection. 500 nm of ure 4 C).
scrambled sequence and PB-19 sequence demonstrated signal This designed E-CRISPR array was evaluated for the
changes of less than 1.5 % and 1.7 %, respectively, which are detection of transforming growth factor b1 (TGF-b1) protein,
lower than the standard error of the signal generated by 1 nm which is a secreted protein contributing to cell proliferation
HPV-16 target, indicating a good selectivity of the Cas12a- and differentiation,[22] and is also recognized as a biomarker
crRNA duplex in differentiating HPV-16 from non-target for hepatocellular carcinoma.[23] The dose-dependent E-
(Figure 3 B). A selectivity test was also performed for PB-19 CRISPR for the detection of TGF-b1 aptamer was first
detection using HPV-16 and scrambled sequence as interfer- evaluated using the previous established trans-cleavage con-
ences (Supporting Information, Figure S7 B), demonstrating ditions (Supporting Information, Figure S8). For proof-of-
the reliable recognition activity of the CRISPR system. concept, a fixed concentration of aptamer was first applied to
Furthermore, as a biosensing platform, discrimination of treat the sample with and without TGF-b1. E-CRISPR was
mismatches in the nucleic acid base pairs is of importance for then applied to analyze the samples with and without TGF-b1
the potential application for the identification of disease demonstrating a clear signal difference (Supporting Informa-
related point mutations.[19] Thus, we next challenged the E- tion, Figure S9). In order to increase the detection resolution
CRISPR with artificial mismatched nucleic acid targets for nanomolar concentration range, a greater degree of
(HPV-16). The recognition mechanism of CRISPR-Cas12a current difference between 1 nm and 50 nm is necessary.
involves the identification of the PAM region on the target to Therefore, a longer trans-cleavage period was investigated to
unwind the DNA target by Cas protein and further hybrid- evaluate whether a higher current difference can be obtained
ization between the crRNA and the target strand.[3a, 14b] as the trans-cleavage activity is a multiple-turnover reactio-
Therefore, we designed mismatches at different positions on n.[3a] Increasing trans-cleavage period indeed leads to a higher
the target (Figure 3 C). E-CRISPR signal was obtained based detection resolution (Supporting Information, Figure S8 B),
on the detection of 1 nm of these artificial targets (Fig- so a trans-cleavage period of 60 min was selected for protein
ure 3 D). Compared with the wild type (WT) HPV-16 detection. Therefore, this strategy might be applied to tune
sequence, mutations in the PAM region and PAM-adjacent the dynamic range and detection limit of the E-CRISPR
region (position 1) led to complete diminishment of the trans- platform, enhancing the detection performance. An aptamer
cleavage signal. This phenomenon indicates the mandatory concentration of 50 nm was selected for protein sample
requirement of the PAM sequence for the Cas12a-crRNA treatment for 30 min. After the treatment, the sample was
duplex to recognize and cleave the target.[20] Moreover, evaluated by E-CRISPR. A linear detection range was
mismatches in the complementary region of crRNA and the achieved covering three order of magnitudes with an LOD
target resulted in decreased trans-cleavage activity, consistent of 0.2 nm (Figure 4 D). The detection specificity was inves-
with previous mismatch tolerance studies using Cas12a.[3a, 21] tigated using conditioned cell medium from human mesen-

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Figure 4. E-CRISPR cascade for protein detection. A) Sample containing protein target of interest is firstly treated by a fixed concentration of
target-specific aptamer (ssDNA). B) A E-CRISPR system is specifically designed for the recognition of the aptamer. The remaining concentration
of aptamer is analyzed by E-CRISPR. C) A representation of SWV results in the presence and absence of the target. D) Linear calibration curve of
TGF-b1 detection with an equation of Y = 0.91X + 1.79 and R-square value of 0.99 (n = 3, SE = 1.54 %). E) Selectivity study through comparison of
the signal outputs in the presence of 10 nm non-target proteins with that obtained in the presence of 10 nm TGF-b1 (n = 3, **P < 0.01 versus
different interference substances). F) Concentration-dependent signals observed within conditioned cell medium harvested at two time-points
during the chondrogenic differentiation program of human mesenchymal stem cells (hMSCs) medium containing TGF-b1. The samples were
analyzed by three sets of individual experiments using three different sensors (n = 3, ***P < 0.05, Day 28 vs. Day 2). The horizontal black dashed
line represents the average signal variation (n = 3) in the presence of conditioned cell medium only.

chymal stem cells (hMSCs) chonodrogenesis, which is a com- probe the CRISPR cleavage activity (E-CRISPR). Owing to
plex matrix, including collagen type II, aggrecan protein, and the high specificity of target recognition, more than a gene
bovine serum albumin. The designed strategy resulted in editing tool, we utilized the CRISPR Type V system, Cas12a
a good selectivity for the target protein over non-specific (cpf1), as an efficient biosensing system, which translates the
molecules, indicating an excellent specificity of the applied target recognition activity into a detectable electrochemical
aptamer in the system (Figure 4 E). We further challenged the signal through an interrogating electrode constructed with
E-CRISPR platform with samples obtained during the non-specific ssDNA. Various factors were investigated to
chondrogenic differentiation of hMSCs, which were cultured produce a high sensitivity E-CRISPR-based detection plat-
in aggregates with complete chondrogenic differentiation form with an optimized on-chip trans-cleavage activity.
medium for 4 weeks.[24] TGF-b1 was produced during the Moreover, our preliminary implementation illustrates that
chondrogenic differentiation process.[25] A clear difference the E-CRIPSR system can be applied not only for nucleic acid
was identified between the conditioned medium obtained at sensing; with the addition of an aptamer-based sensing
day 2 and day 28 (Figure 4 F). These results are in agreement cascade, the E-CRISPR can also be utilized for protein
with the transcriptome analyses of the same samples per- detection, providing a generalizable, robust, and cost-effec-
formed during chondrogenesis of hMSCs (Supporting Infor- tive detection system.
mation, Figure S10), indicating a reliable performance of the
designed E-CRISPR array for protein detection. The nucleic-
acid-based receptor is a generalized recognition element for Acknowledgements
both protein and small molecule.[26] Hence, the designed E-
CRISPR array can also be extended to a wide variety of The authors acknowledge the staff of the Electronics Design
analytes. Center for the experimental support and Xintong Cao for the
artwork. We acknowledge the funding supports from National
Institute of Health under the grant number, NIBIB:1-
Conclusion P41EB021911 and R01DK113185. We also acknowledge the
funding supports from Wallace R. Persons Research Fund
This study introduces a new strategy for the development from Case Alumni Association.
of electrochemical biosensors by using electrochemistry to

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[10] G. Maule, A. Casini, C. Montagna, A. S. Ramalho, K. De Boeck,
Keywords: bioanalytical chemistry · biosensor · Z. Debyser, M. S. Carlon, G. Petris, A. Cereseto, Nat. Commun.
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Franceschi, P. E. Castle, J. Walker, R. Zuna, A. R. Kreimer, Version of record online: && &&, &&&&

&&&& www.angewandte.org  2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2019, 58, 2 – 9
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These are not the final page numbers!

 Angewandte
Research Articles Chemie

Research Articles
Biosensors An electrochemical biosensor based on
CRISPR-Cas12a (cpf1), termed E-CRISPR,
Y. Dai,* R. Somoza, L. Wang, J. F. Welter, is reported. Utilizing the trans-cleavage
Y. Li, A. I Caplan,* activity of Cas12a, E-CRISPR delivers
C. C. Liu* &&&&—&&&& a cost-effective and portable biosensing
platform for the detection of nucleic acids
Exploring the Trans-Cleavage Activity of and proteins.
CRISPR-Cas12a (cpf1) for the
Development of a Universal
Electrochemical Biosensor

Angew. Chem. Int. Ed. 2019, 58, 2 – 9  2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org &&&&
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These are not the final page numbers!