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Scalable biological signal recording in mammalian cells using Cas12a base editors

Nature Chemical Biology volume 18, pages 742–750 (2022)Cite this article

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Abstract

Biological signal recording enables the study of molecular inputs experienced throughout cellular history. However, current methods are limited in their ability to scale up beyond a single signal in mammalian contexts. Here, we develop an approach using a hyper-efficient dCas12a base editor for multi-signal parallel recording in human cells. We link signals of interest to expression of guide RNAs to catalyze specific nucleotide conversions as a permanent record, enabled by Cas12’s guide-processing abilities. We show this approach is plug-and-play with diverse biologically relevant inputs and extend it for more sophisticated applications, including recording of time-delimited events and history of chimeric antigen receptor T cells’ antigen exposure. We also demonstrate efficient recording of up to four signals in parallel on an endogenous safe-harbor locus. This work provides a versatile platform for scalable recording of signals of interest for a variety of biological applications.

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Fig. 1: Base editing with hyperCas12a as a platform for signal recording.
Fig. 2: Modular recording of diverse biological signals.
Fig. 3: Recording of analog signal characteristics.
Fig. 4: Temporal resolution of cellular history within recording window.
Fig. 5: Dual-function effector and recorder cells.
Fig. 6: Simultaneous recording of multiple signals in parallel.

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Data availability

All data supporting main figures and extended data figures have been included in source data files. Plasmids are available from Addgene with the following accession codes: 183098, 183203, 183204, 183205, 183206, 183207, 183623, 183624, 183625, 183626, 183627, 183628 and 183629. Raw deep sequencing data are available at National Center for Biotechnology Information Bioproject PRJNA818698. No custom code was used in this study. Source data are provided with this paper.

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Acknowledgements

We thank members of the Qi laboratory, including M. Chavez, V. Tieu, P. Finn, T. Abbott, A. Chemparathy, X. Xu, J. Bian and M. Nakamura, as well as E. Gonzalez Diaz and F. Yang for advice and helpful discussions. The authors also thank the Stanford Shared Protein and Nucleic Acid Facility for technical support. H.R.K. acknowledges support from the National Science Foundation Graduate Research Fellowship Program, Stanford Bio-X Fellowship Program and Siebel Scholars Foundation. L.S.Q. acknowledges support from the Li Ka Shing Foundation, the Stanford Maternal and Child Health Research Institute through the Uytengsu-Hamilton 22q11 Neuropsychiatry Research Award Program, National Science Foundation CAREER award (award no. 2046650) and California Institute for Regenerative Medicine (CIRM, DISC2-12669). L.S.Q. is a Chan Zuckerberg Biohub investigator. The work is supported by National Science Foundation CAREER award (award no. 2046650) and the Li Ka Shing Foundation.

Author information

Authors and Affiliations

  1. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Hannah R. Kempton, Kasey S. Love, Lucie Y. Guo & Lei S. Qi

  2. Department of Ophthalmology, Stanford University, Stanford, CA, USA

    Lucie Y. Guo

  3. Sarafan ChEM-H, Stanford University, Stanford, CA, USA

    Lei S. Qi

  4. Chan Zuckerberg BioHub, San Francisco, CA, USA

    Lei S. Qi

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  1. Hannah R. Kempton
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Contributions

H.R.K. and L.S.Q. conceived the idea. H.R.K. designed and performed experiments for all figures. K.S.L. designed constructs and performed experiments for initial testing of Cas12 base editor constructs. L.Y.G. designed constructs and contributed for testing of hyperCas12 mutants. H.R.K. analyzed the data. H.R.K. and L.S.Q. wrote the manuscript. All authors read and commented on the manuscript.

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Correspondence to Lei S. Qi.

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Competing interests

The authors have filed a provisional patent via Stanford University partly related to the work (US patent no. 63/148,652; international application patent no. PCT/US2022/016223). L.S.Q. is a founder of Epicrispr Biotechnologies.

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Extended data

Extended Data Fig. 1 Base editing with hyperCas12a for signal recording.

a, Left, representative flow cytometry histograms showing GFP fluorescence in HEK cells 3 days post-transfection with GFP* reporter, U6-driven guide and wild type (WT) or mutant (hyper) dCas12a-ABE. crLacZ, non-targeting control. Right, quantification of percentage GFP + cells for 3 independent replicates. To go with Fig. 1c. b, Representative flow cytometry histograms for GFP* HEK cells transfected with the dox-recording constructs, 3 days post-transfection and stimulation with dox. To go with Fig. 1f. c, Representative flow cytometry histograms for GFP* HEK cells transfected with hyperdCas12a base editor and NFKB-driven crGFP*, 3 days post-transfection and stimulation with TNFα. To go with Fig. 1g.

Source data

Extended Data Fig. 2 Optimizing inducible crRNAs for lentiviral transduction.

a, Left, designs tested for transferring the inducible guide cassette into a lentiviral backbone. The inducible construct was placed in reverse orientation, to prevent the polyA from disrupting lentiviral packaging. Right, quantification of reporter GFP fluorescence in HEK cells transiently transfected with inducible guide, mutant base editor, and reporter, as well as additional rtTA for design 2. Data shown for 2 independent replicates, 3 days post-transfection and stimulation with dox. Design 1 was selected moving forward, as despite slightly worse performance than design 2 it had benefit of including constitutive open reading frame for selection. b, Left, schematic showing incorporation of additional repeat-spacer cassette to increase amount of guide available per mRNA transcript. Right, GFP fluorescence in GFP* HEK cells stably transduced with dox-inducible guide, 3 days post-transfection with base editor and stimulation with dox. Data shown for 3 independent replicates. c, Left, representative histogram of GFP* HEK cells stably transduced with NFKB-crRNA, 3 days post-transfection with base editor and stimulation with TNFα. Right, quantification of mean fluorescence and percent GFP + for 3 independent replicates. d, Quantification of A→G mutations in stably transduced NFKB-crRNA cells at the targeted stop codon in the GFP* locus as measured by MiSeq. Data are shown for 3 independent replicates.

Source data

Extended Data Fig. 3 Dual signal recording using the GFP** reporter.

a, Schematic showing the GFP** dual recording reporter, which expresses GFP upon the activity of two distinct GFP* guides removing two stop codons. b, Left, representative histograms of HEK cells 3 days post-transfection with GFP**, base editor, and combinations of U6-crGFP*. Right, quantification of mean GFP for 3 independent replicates. c, Modified lentiviral constructs used to transduce two inducible guides into cells and select with a single antibiotic, through use of split hygromycin. d, Representative histograms of GFP** HEK cells stably transduced with NFKB and TRE3G inducible guides shown in c and transfected with base editor, 3 days posttransfection and stimulation with TNFα/dox. e, Quantification of mean fluorescence (left) and percentage GFP + (right) of GFP** HEK cells stably transduced with NFKB and TRE3G inducible guides and transfected with base editor, 3 days post transfection and stimulation with TNFα/dox. All data shown for 3 independent replicates. f, Quantification of A→G mutations at each targeted stop codon in the GFP** locus as measured by MiSeq. Data are shown for 3 independent replicates.

Source data

Extended Data Fig. 4 Modular recording of diverse biological signals.

a, Schematic showing modular recording by incorporating different inducible promoters into the guide construct. b, Cellular memory of NFAT signaling, stimulated by PMA. c, Cellular memory of oxidative stress, stimulated by tBHQ. ROS = reactive oxygen species. All experiments in b-c shown in GFP* HEK cells stably transduced with respective inducible guide, for 3 independent replicates 3 days post transfection with base editor.

Source data

Extended Data Fig. 5 Recording memory of antigen encounter.

a, Constructs used for SynNotch-induced guide expression. b, Cellular memory of antigen encounter via SynNotch, in GFP* HEK cells transfected with guide, SynNotch, and base editor, then cocultured with -/+CD19 HEK cells. Data shown for 3 independent replicates c, Constructs used for recording CD19-CAR activation in Jurkat cells. The top two constructs were stably integrated, and the bottom two were electroporated. d, Representative flow cytometry histograms for signal recording of CD19 antigen encounter in Jurkat cells. Histograms show BFP (induced by NFAT promoter) vs GFP (from successful base editing event of GFP* reporter) in engineered Jurkat CAR-T cells, after 48 hours of co-culture with K562 cells -/+CD19. To go with Fig. 2d.

Source data

Extended Data Fig. 6 Characterization of recording parameters.

a, GFP* HEK cells stably transduced with lentiviral NFKBp-guide and transfected with base editor were stimulated with 100 ng/mL TNFα for 3 days, then analyzed via flow cytometry. Randomly selected sub-populations of cells of the indicated size were analyzed using downsampling. Statistical analysis was performed using a two-sided t test without adjustment for multiple comparisons. Data shown for 3 independent replicates. To go with Fig. 3a. b, The mean GFP fluorescence for each downsampled cell population for the NFKB-recording circuit, +TNFα condition is plotted against the number of cells analyzed, to go with Fig. 3a. Dotted lines with gray shading indicate standard deviation of 3 independent replicates. c, Dose response for GFP* HEK cells stably transduced with lentiviral NFKBp-guide and transfected with base editor. Mean BFP fluorescence is shown 3 days post-transfection and stimulation with TNFα. Error bars indicate standard deviation of 3 independent replicates. To go with Fig. 3b. d, Mean BFP (expressed under NFKB promoter) plotted against mean GFP (“memory” reporter) for TNFα dose-response experiment. Data were fitted using a linear regression. Error bars indicate standard deviation of 3 independent replicates. e, Experimental timeline used for testing different durations of TNFα stimulation.

Source data

Extended Data Fig. 7 Recording history of NFKB signaling at the AAVS1 locus.

a, Left, constructs used for signal recording using inducible AAVS1 targeting guides in HEK cells. Right, experimental timeline used for genomic DNA (gDNA) collection. b, Single-input recording at the AAVS1 locus in HEK cells transfected with base editor and NFKB-driven crAAVS1_4, and stimulated with TNFα for 3 days. The %A→G conversion as measured by MiSeq is shown for A11 (left) and A14 (right), with number indicating position of A relative to PAM. Data shown for 3 independent replicates. c, Left, constructs used for recording at the AAVS1 locus in Jurkat cells. Right, experimental timeline. d, Recording of NFKB-mediated inflammation in Jurkat cells at the AAVS1 locus, as measured by MiSeq. Data shown for 3 independent replicates.

Source data

Extended Data Fig. 8 Utilizing two parallel guides for more detailed record of input signal characteristics.

a, Schematic showing two input signals with distinctive patterns but the same area under the curve. b, Promoter engineering strategy used to reduce the sensitivity of NFKB-responsive promoter, to generate two inducible guide constructs that respond differently to input TNFα signal. c, Mean BFP fluorescence of HEK cells 3 days post-transfection with NFKB promoter variants and stimulation with TNFα. Left, raw mean BFP values are plotted. Right, mean BFP values normalized to the maximum of each respective promoter are plotted. Error bars indicate standard deviation of 3 independent replicates. d, Quantification of %A→G conversion as measured by MiSeq for HEK cells transfected with base editor, 5X-NFKB-crAAVS1_4, and 2X-NFKB-crAAVS1_8. One day after transfection, cells were stimulated with either 100 ng/mL TNFα for 0.25 hours, or 0.5 ng/mL TNFα for 50 hours. All data shown for 3 independent replicates. Statistical analysis was performed using a two-sided t test without adjustment for multiple comparisons.

Source data

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Kempton, H.R., Love, K.S., Guo, L.Y. et al. Scalable biological signal recording in mammalian cells using Cas12a base editors. Nat Chem Biol 18, 742–750 (2022). https://doi.org/10.1038/s41589-022-01034-2

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