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CRISPR-Mediated Isogenic Cell-SELEX Approach for Generating Highly Specific Aptamers Against Native Membrane Proteins

  • 2020 CMBE Young Innovators Issue
  • Published:
Cellular and Molecular Bioengineering Aims and scope Submit manuscript

Abstract

Introduction

The generation of affinity reagents that bind native membrane proteins with high specificity remains challenging. Most in vitro selection paradigms utilize different cell types for positive and negative rounds of selection (where the positive selection is against a cell that expresses the desired membrane protein and the negative selection is against a cell that lacks the protein). However, this strategy can yield affinity reagents that bind unintended membrane proteins on the target cells. To address this issue, we developed a systematic evolution of ligands by exponential enrichment (SELEX) scheme that utilizes isogenic pairs of cells generated via CRISPR techniques.

Methods

Using a Caco-2 epithelial cell line with constitutive Cas9 expression, we knocked out the SLC2A1 gene (encoding the GLUT1 glucose transporter) via lipofection with synthetic gRNAs. Cell-SELEX rounds were carried out against wild-type and GLUT1-null cells using a single-strand DNA (ssDNA) library. Next-generation sequencing (NGS) was used to quantify enrichment of prospective binders to the wild-type cells.

Results

10 rounds of cell-SELEX were conducted via simultaneous exposure of ssDNA pools to wild-type and GLUT1-null Caco-2 cells under continuous perfusion. The top binders identified from NGS were validated by flow cytometry and immunostaining for their specificity to the GLUT1 receptor.

Conclusions

Our data indicate that highly specific aptamers can be isolated with a SELEX strategy that utilizes isogenic cell lines. This approach may be broadly useful for generating affinity reagents that selectively bind to membrane proteins in their native conformations on the cell surface.

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Abbreviations

SELEX:

Systematic evolution of ligands through exponential enrichment

CRISPR:

Clustered regularly interspaced short palindromic repeats

GLUT:

Glucose transporter

TIDE:

Tracking of indels by decomposition

NGS:

Next-generation sequencing

iPSC:

Induced pluripotent stem cell

BMEC:

Brain microvascular endothelial cells

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Acknowledgments

We gratefully acknowledge Allison Bosworth for providing iPSC-derived BMECs, Ella Hoogenboezem for providing MDA-MB-231 cells, and Everett Allchin for culturing HEK-293 cells. This research was supported by a Ben Barres Early Career Acceleration Award from the Chan Zuckerberg Initiative (Grant 2018-191850 to ESL), Grant A20170945 from the BrightFocus Foundation (ESL), Grant IRG-58-009-56 from the American Cancer Society (ESL), and an Engineering Immunity Pilot Grant from Vanderbilt University (ESL). This work was further supported by facilities at Vanderbilt University, including the Vanderbilt University Medical Center Flow Cytometry shared resource (supported by the Vanderbilt Ingram Cancer Center NIH Grant P30 CA68485 and the Vanderbilt Digestive Disease Research Center NIH Grant DK058404) and the Vanderbilt Technologies for Advanced Genomics core facility (supported by the Vanderbilt Ingram Cancer Center NIH Grant P30 CA68485, the Vanderbilt Vision Center NIH Grant P30 EY08126, and NIH/NCRR Grant G20 RR030956). EHN is supported by a Graduate Research Fellowship from the National Science Foundation (DGE-1445197). DAB was supported by the Vanderbilt University Medical Scientist Training Program (T32 GM007347). We thank the Cooperative Human Tissue Network, an NIH/NCI sponsored resource, for providing human brain tissue.

Conflict of interest

The authors declare no conflicts of interest.

Ethical Approval

All human tissue used in this study was de-identified and did not require IRB approval.

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Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, Vanderbilt University, PMB 351604, 2301 Vanderbilt Place, Nashville, TN, 37235-1604, USA

    Jonah C. Rosch, Emma H. Neal, Mohsin Rahim & Ethan S. Lippmann

  2. Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA

    Daniel A. Balikov & Ethan S. Lippmann

  3. Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, MI, USA

    Daniel A. Balikov

  4. Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville, TN, USA

    Ethan S. Lippmann

  5. Interdisciplinary Materials Science Program, Vanderbilt University, Nashville, TN, USA

    Ethan S. Lippmann

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  1. Jonah C. Rosch
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Correspondence to Ethan S. Lippmann.

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Rosch, J.C., Neal, E.H., Balikov, D.A. et al. CRISPR-Mediated Isogenic Cell-SELEX Approach for Generating Highly Specific Aptamers Against Native Membrane Proteins. Cel. Mol. Bioeng. 13, 559–574 (2020). https://doi.org/10.1007/s12195-020-00651-y

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