CRISPR-enabled point-of-care genotyping for APOL1 genetic risk assessment

Robert Greensmith; Isadora T Lape; Cristian V Riella; Alexander J Schubert; Jakob J Metzger; Anand S Dighe; Xiao Tan; Bernhard Hemmer; Josefine Rau; Sarah Wendlinger; Nora Diederich; Anja Schütz; Leonardo V Riella; Michael M Kaminski

doi:10.1038/s44321-024-00126-x

CRISPR-enabled point-of-care genotyping for APOL1 genetic risk assessment

EMBO Mol Med. 2024 Oct;16(10):2619-2637. doi: 10.1038/s44321-024-00126-x. Epub 2024 Sep 13.

Authors

Robert Greensmith^{1

2}, Isadora T Lape³, Cristian V Riella^{4

5}, Alexander J Schubert^{1

2

6}, Jakob J Metzger¹, Anand S Dighe^{5

7}, Xiao Tan^{8

9

10

11}, Bernhard Hemmer^{12

13}, Josefine Rau¹, Sarah Wendlinger^{1

2}, Nora Diederich^{1

2}, Anja Schütz¹⁴, Leonardo V Riella^{15

16

17}, Michael M Kaminski^{18

19

20}

Affiliations

¹ Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany.
² Department of Nephrology and Medical Intensive Care, Charité Universitätsmedizin Berlin, Berlin, Germany.
³ Center for Transplantation Sciences, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA.
⁴ Nephrology Division, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA, 02215, USA.
⁵ Harvard Medical School, Boston, MA, 02115, USA.
⁶ Berlin Institute of Health, Berlin, Germany.
⁷ Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.
⁸ Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.
⁹ Division of Gastroenterology, Massachusetts General Hospital, Boston, MA, USA.
¹⁰ Institute for Medical Engineering and Science and Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
¹¹ Infectious Disease and Microbiome Program, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
¹² Department of Neurology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
¹³ Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
¹⁴ Protein Production & Characterization, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, 13125, Berlin, Germany.
¹⁵ Center for Transplantation Sciences, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA. [email protected].
¹⁶ Department of Surgery, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA. [email protected].
¹⁷ Division of Nephrology, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA. [email protected].
¹⁸ Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany. [email protected].
¹⁹ Department of Nephrology and Medical Intensive Care, Charité Universitätsmedizin Berlin, Berlin, Germany. [email protected].
²⁰ Berlin Institute of Health, Berlin, Germany. [email protected].

Abstract

Detecting genetic variants enables risk factor identification, disease screening, and initiation of preventative therapeutics. However, current methods, relying on hybridization or sequencing, are unsuitable for point-of-care settings. In contrast, CRISPR-based-diagnostics offer high sensitivity and specificity for point-of-care applications. While these methods have predominantly been used for pathogen sensing, their utilization for genotyping is limited. Here, we report a multiplexed CRISPR-based genotyping assay using LwaCas13a, PsmCas13b, and LbaCas12a, enabling the simultaneous detection of six genotypes. We applied this assay to identify genetic variants in the APOL1 gene prevalent among African Americans, which are associated with an 8-30-fold increase in the risk of developing kidney disease. Machine learning facilitated robust analysis across a multicenter clinical cohort of more than 100 patients, accurately identifying their genotypes. In addition, we optimized the readout using a multi-analyte lateral-flow assay demonstrating the ability for simplified genotype determination of clinical samples. Our CRISPR-based genotyping assay enables cost-effective point-of-care genetic variant detection due to its simplicity, versatility, and fast readout.

Keywords: APOL1; Biomarkers; CRISPR-based-genotyping; Diagnostics; Kidney Disease.

MeSH terms

Apolipoprotein L1* / genetics
Black or African American / genetics
CRISPR-Cas Systems / genetics
Clustered Regularly Interspaced Short Palindromic Repeats / genetics
Genetic Predisposition to Disease
Genotype
Genotyping Techniques / methods
Humans
Kidney Diseases / diagnosis
Kidney Diseases / genetics
Point-of-Care Systems*
Risk Assessment / methods

Substances

Apolipoprotein L1
APOL1 protein, human

Grants and funding

KA5060/1-1/Deutsche Forschungsgemeinschaft (DFG)