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Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection

Nature Chemical Biology volume 19, pages 45–54 (2023)Cite this article

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Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 13 (Cas13) has been rapidly developed for nucleic-acid-based diagnostics by using its characteristic collateral activity. Despite the recent progress in optimizing the Cas13 system for the detection of nucleic acids, engineering Cas13 protein with enhanced collateral activity has been challenging, mostly because of its complex structural dynamics. Here we successfully employed a novel strategy to engineer the Leptotrichia wadei (Lwa)Cas13a by inserting different RNA-binding domains into a unique active-site-proximal loop within its higher eukaryotes and prokaryotes nucleotide-binding domain. Two LwaCas13a variants showed enhanced collateral activity and improved sensitivity over the wild type in various buffer conditions. By combining with an electrochemical method, our variants detected the SARS-CoV-2 genome at attomolar concentrations from both inactive viral and unextracted clinical samples, without target preamplification. Our engineered LwaCas13a enzymes with enhanced collateral activity are ready to be integrated into other Cas13a-based platforms for ultrasensitive detection of nucleic acids.

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Fig. 1: Design of N- and C-terminal RBD fusions to LwaCas13a.
Fig. 2: Structure-guided design of RBD–LwaCas13a fusion proteins with enhanced collateral activity.
Fig. 3: Optimizing reporter length for enhanced collateral activity.
Fig. 4: RBD#3L and #4L enhance the collateral activity in a spacer-independent manner and maintain the specificity for target recognition.
Fig. 5: Improved detection of various spiked-in targets.
Fig. 6: RBD fusions facilitate electrochemical detection of RNA at attomolar sensitivity.

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Data availability

All data generated or analyzed during this study are included in this published article and its Supplementary Information. The previous structural data that support the findings of this study are available in the PDB (IDs: 5XWP, 5MPL, and 2MXY). Source data are provided with this paper.

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Acknowledgements

We thank P. Lillehoj for proofreading the manuscript. We acknowledge funding from NSF CBET-2031242, Welch Foundation (C-1952), and Rice University Startup fund (to X.G.); the University of Connecticut Startup fund and NSF CBET-2103025 (to Y.Z.); and the Welch Foundation (C-2033-20200401) and the Cancer Prevention & Research Institute of Texas (CPRIT) Award RR190046 (to Y.G.).

Author information

Author notes

  1. These authors contributed equally: Jie Yang, Yang Song.

Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX, USA

    Jie Yang, Jeffrey A. Vanegas, Zheng You & Xue Gao

  2. Department of Biomedical Engineering, University of Connecticut, Storrs, CT, USA

    Yang Song, Yuxuan Zhang, Zhengyan Weng & Yi Zhang

  3. Institute of Materials Science, University of Connecticut, Storrs, CT, USA

    Yang Song, Yuxuan Zhang & Zhengyan Weng

  4. Department of Biosciences, Rice University, Houston, TX, USA

    Xiangyu Deng, Advaith Peddi & Yang Gao

  5. Department of Pathology and Laboratory Medicine, UConn Health, Farmington, CT, USA

    Lori Avery

  6. Division of Infectious Diseases, Department of Medicine, UConn Health, Farmington, CT, USA

    Kevin D. Dieckhaus

  7. Polymer Program, Institute of Materials Science, University of Connecticut, Storrs, CT, USA

    Yi Zhang

  8. Department of Bioengineering, Rice University, Houston, TX, USA

    Xue Gao

  9. Department of Chemistry, Rice University, Houston, TX, USA

    Xue Gao

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Contributions

X.G. designed and supervised the research. J.Y., Y.G., and X.G. conceived and designed the RBD fusion protein engineering. Y.G. performed the structural analysis. J.Y. and X.D. expressed and purified proteins, and transcribed and purified RNAs. J.Y. performed the experiments and analyzed the data related to nuclease activity fluorescence plate reader assay, kinetic analysis, and gel assay, with the assistance of J.A.V. and Z.Y. X.D. performed gel shift assays and analyzed the data. Yi.Z. and Y.S. conceived and designed the Cas13a–electrochemical system. Y.S. and Z.W. performed the experiments related to the Cas13a–electrochemical system, and J.Y., Y.S., and Z.W. analyzed the data. L.A. and K.D.D. provided and verified the clinical samples. Y.S. and Yu.Z. performed RT–qPCR and analyzed the data. J.Y. performed and analyzed results from all other experiments with assistance from J.A.V., Z.Y. and A.P. J.Y., Y.S., and X.G. drafted the manuscript with input from all authors.

Corresponding authors

Correspondence to Yang Gao, Yi Zhang or Xue Gao.

Ethics declarations

Competing interests

X.G., J.Y., and Y.G. are co-inventors on a provisional patent application 63/285,304 (filed) relating to the engineered Cas13 variants described in this manuscript. The remaining authors declare no competing interests.

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Nature Chemical Biology thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Time course of N- and C- terminal RBD fusions.

a and b, Background-subtracted fluorescence in the detection of 10 pM synthetic SARS-CoV-2 N gene fragment targets (T1) using N- (a) and C- (b) terminal RBD fusions, expressed as mean ± s.e.m. from four technical replicates.

Source data

Extended Data Fig. 2 Testing of RBD loop-fusion variants.

Each of the fusion proteins was assembled with the crRNA as described in Methods. a–c, Testing of seven candidate RBDs (RBD#1–RBD#7) fused to LwaCas13a. a and b, Raw fluorescence over a course of 120 min in the reactions with or without 10 pM synthetic RNA targets: a, SARS-CoV-2 RdRp gene fragment (T2); b, ssRNA1 (T3). NTC, non-target control. c, The 120-min background-subtracted fluorescence from a and b. d–f, Testing of RBD#3 and RBD#4 fused to various positions on Loop 1 of LwaCas13a. The six variants were RBD#3 and #4 inserted after N415, N416, and K417, respectively. d and e, Raw fluorescence data over a course of 120 min in the reactions with or without 10 pM synthetic RNA targets: d, SARS-CoV-2 RdRp gene fragment (T2); e, ssRNA1 (T3). f, The 30-min background-subtracted fluorescence from d and e. g and h, Testing of tandem RBD insertions. The four variants for comparison were RBD#3, RBD#4, RBD#3–linker–RBD#4, and RBD#4–linker–RBD#3 inserted after N415 on Loop 1 of LwaCas13a, respectively. A 10-aa flexible linker (SGGSGGSGGS) was used to connect two RBDs in tandem. g, Background-subtracted fluorescence data over a course of 60 min in the reactions with 10 pM SARS-CoV-2 N gene fragment synthetic RNA targets (T1). h, The 30-min and 60-min background-subtracted fluorescence from g. a, b, d, e, and g, Lines and error bars represented mean ± s.e.m. from four technical replicates; c, f, and h, bar plots were expressed as mean ± s.e.m. from four technical replicates. Two-tailed p values were calculated using unpaired t-tests with Welch’s correction: ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (see SourceData for p value).

Source data

Extended Data Fig. 3 Optimizing reporter length for enhanced collateral activity.

a and b, Representative gel images of the cleavage of 5′-FAM-labeled U5, U11, U15, and U20 reporters by WT LwaCas13a, RBD #3L, and RBD#4L over 0–30 min. RBD fusions and WT were tested side-by-side. c–e, Quantified percentage of cleaved products as shown in a and b, represented by mean ± s.e.m. of three technical replicates from two independent experiments. Gels were analyzed by ImageJ. The percentage of cleaved products was calculated by the intensity of product bands divided by the summed intensity of the product and substrate bands within one lane and normalized to the reporter-only control.

Source data

Extended Data Fig. 4 LoD analysis of RBD#3L, RBD#4L, and WT LwaCas13a in reactions supplemented with BSA and Triton X-100.

a–c, Raw fluorescence data measured for 120 min, expressed as mean ± s.e.m. from four technical replicates. RBD#3L, RBD#4L and WT LwaCas13a were complexed with crRNA (cr1) and tested with 2.5, 1, 0.25, 0.1, 0.025 pM of the synthetic RNA (T1) in reaction buffer supplemented with 100 μg/mL BSA and 0.01% Triton X-100 (as in Supplementary Fig. 2b). The 0 pM target samples were used as non-target control (NTC). d, The bar plot represented the 120-min background-subtracted fluorescence expressed as mean ± s.e.m. from four technical replicates. Adjusted p values were calculated using two-way ANOVA with Dunnett’s test: **p = 0.0059, ***p = 0.0007, ****p < 0.0001, compared to background signal (non-target control) (see SourceData for adjusted p value). e, The velocities of each reaction within 60 min were obtained by linear regression from a–c and were plotted as functions of the target concentration. Outliers were identified by the ROUT method with a Q = 5%. Analytical LoD of three proteins was listed in Supplementary Table 2.

Source data

Extended Data Fig. 5 Quantification of RNA binding with RBD#3L, RBD#4L, and WT LwaCas13a.

a and c, Representative EMSA gel images of inactive HEPN2 mutants (R1046A/H1051A), that is, dRBD#3L, dRBD#4L, and dWT complexed with crRNA binding to 5 nM of body-radiolabeled (32P) target RNA. (a, T26; c, T1). The protein:crRNA complex with a molar ratio of 1:0.95 was serially diluted to 288, 192, 128, 86, 57, 38, 25, 17, 12, 8 and 5 nM. The 0-nM control was target RNA with reaction buffer. b and d, Calculation of binding affinity between target RNA and RNP. Bound and unbound fractions from a and c were quantified by densitometry and fitted to standard binding isoforms. Mean from two technical replicates was plotted. e, Representative EMSA gel images of protein:crRNA:target ternary complex binding with 100 nM of 5'-FAM-labeled U20 reporter. The dRBD#3L, dRBD#4L and dWT complex with crRNA with a molar ratio of 2:1 was serially diluted to 2, 1.8, 1.6, 1.4, 1.2, 1, 0.8, 0.4, 0.2, 0.1 and 0.05 μM. An equal amount of 50 pM non-labeled target (T1) was added to the RNPs to mimic the target-bound ternary complex. The 0-μM control was the reporter with reaction buffer. f, Calculation of binding affinity between reporter RNA and target-bound RNP. Bound and unbound fractions from e were quantified by densitometry and fitted to standard binding isoforms. Mean from two technical replicates was plotted. Means of the dissociation constant with 95% CI are listed in Supplementary Table 3.

Source data

Extended Data Fig. 6 Kinetic analysis of RBD#3L, RBD#4L and WT LwaCas13a.

a–c, Progress curves of cleaved reporters versus time. Data were plotted as mean ± s.e.m. from two independent experiments each with four technical replicates. The RNP complex of RBD#3L, RBD#4L, and WT was incubated with 50 pM of the targets (T1) for 10 min at 37 °C prior to cleavage reaction using 6400, 3200, 1600, 800, 400, 200, 100 and 50 nM of the U11 substrates. The initial velocities (within 600 s) of each reaction were obtained by linear regression. d, Initial velocities from two independent experiments each with four technical replicates obtained from a–c were plotted as mean ± s.e.m. versus the U11 reporter concentration and fitted to a Michaelis-Menten curve. Summary of kinetic parameters are listed in Supplementary Table 4.

Source data

Extended Data Fig. 7 Testing RBD#3L, #4L, and WT LwaCas13a in the detection of viral RNA and microRNA spiked-in samples.

Background-subtracted fluorescence over 120 min in the detection of a, b, 10 pM of SARS-CoV-2 N gene synthetic targets spiked into 8% VTM, 8% saliva, or reaction buffer; c, 10 pM of Zika synthetic targets spiked into 8% urine, or reaction buffer, d, 10 pM of Dengue synthetic targets spiked into 2.5% serum, or reaction buffer; e, 10 pM of Ebola synthetic targets spiked into 2.5% plasma, or reaction buffer; f, g, 10 pM of miR-19b spiked into 2.5% plasma, 2.5% serum, or reaction buffer; h, i, 200 pM of miR-2392 spiked into 2.5% serum, 8% urine, or reaction buffer; j, 10 pM of miR-2392 in reaction buffer. All reactions were supplemented with 50 ng total RNA extracted from HEK293T cells but j. Background-subtracted fluorescence data were expressed as mean ± s.e.m. from four technical replicates (a–i), or 2 independent experiments, each with four technical replicates (j).

Source data

Extended Data Fig. 8 Testing RBD#3L and WT LwaCas13a in the detection of viral RNA spiked-in samples.

Background-subtracted fluorescence over 120 min in the detection of a, 50 pM SARS-CoV-2 N gene synthetic targets (T1) spiked into 16% VTM, 8% VTM, or reaction buffer; b, 50 pM of Zika synthetic targets (T27) spiked into 16% urine, 8% urine, or reaction buffer, c, 50 pM of Dengue synthetic targets (T28) spiked into 4.8% serum, 2.5% serum, or reaction buffer; d, 50 pM of Ebola synthetic targets (T29) spiked into 4.8% plasma, 2.5% plasma, or reaction buffer. All reactions were supplemented with 50 ng total RNA extracted from HEK293T cells as background. Background-subtracted fluorescence data were expressed as mean ± s.e.m. from four technical replicates.

Source data

Extended Data Fig. 9 Theoretical LoD of the WT LwaCas13a and RBD#3L coupled to the electrochemical sensor in the detection of synthetic RNA target.

a, ΔI% (mean ± s.d. from three technical replicates) from Fig. 6a was plotted as a function of target concentration (0, 0.01, 0.1, 0.5, and 1 pM) and fitted by linear regression with replicate y values considered as individual data points. b, ΔI% (mean ± s.d. from three technical replicates) from Fig. 6b was plotted as a function of target concentration (0, 1, 10, 50, and 100 aM) and fitted by linear regression. c, ΔI% measurement from samples with activated RBD#3L RNP. The sample treatment and assay conditions were in parallel with Fig. 6b except the reaction buffer used was Tris (Supplementary Table 7). ΔI% (mean ± s.d. from three technical replicates) was plotted as a function of target concentration (0, 1, 5, 10, and 20 aM) and fit by linear regression. LoD values were 0.79 pM for WT, 19.76 aM for RBD#3L in HEPES buffer, and 3.28 aM for RBD#3L in Tris buffer, respectively.

Source data

Supplementary information

Supplementary Information

Supplementary Figs 1–11, Supplementary Tables 1–7, Supplementary Notes 1–6, uncropped gel images, Supplementary References.

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Supplementary Data 1

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Yang, J., Song, Y., Deng, X. et al. Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection. Nat Chem Biol 19, 45–54 (2023). https://doi.org/10.1038/s41589-022-01135-y

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