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Cited by 163CRISPR and Genetic EngineeringGenomics and Rare DiseasesLysosomal Storage Disorders Research

Saturation variant interpretation using CRISPR prime editing

Open access
Erwood, Steven, Bily, Teija M. I., Lequyer, Jason, Yan, Joyce, Gulati, Nitya, Brewer, Reid A., Zhou, Liangchi, Pelletier, Laurence +2 more·2022·Nature Biotechnology
DOI 10.1038/s41587-021-01201-1

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Abstract

High-throughput functional characterization of genetic variants in their endogenous locus has so far been possible only with methods that rely on homology-directed repair, which are limited by low editing efficiencies. Here, we adapted CRISPR prime editing for high-throughput variant classification and combined it with a strategy that allows for haploidization of any locus, which simplifies variant interpretation. We demonstrate the utility of saturation prime editing (SPE) by applying it to the NPC intracellular cholesterol transporter 1 gene (NPC1), mutations in which cause the lysosomal storage disorder Niemann-Pick disease type C. Our data suggest that NPC1 is very sensitive to genetic perturbation, with 410 of 706 assayed missense mutations being classified as deleterious, and that the derived function score of variants is reflective of diverse molecular defects. We further applied our approach to the BRCA2 gene, demonstrating that SPE is translatable to other genes with an appropriate cellular assay. In sum, we show that SPE allows for efficient, accurate functional characterization of genetic variants.

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  • Each pegRNA directs editing to the target locus and provides the reverse-transcription template needed to install the programmed variant at the endogenous site.

    “Each pegRNA directs editing to the target locus and provides the reverse-transcription template needed to install the programmed variant at the endogenous site (56, 57) (Fig. 2B).”

    From Massively parallel functional genomic assays in endocrinology: from promise to delivery (2026) · cites this paper as [57]

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Research works printed but not linked: 45

  1. Cooper, G. M. Parlez-vous VUS? Genome Res 25, 1423-1426, doi:10.1101/gr.190116.115 (2015). · DOI
  2. Starita, L. M. et al. Variant Interpretation: Functional Assays to the Rescue. Am J Hum Genet 101, 315-325, doi:10.1016/j.ajhg.2017.07.014 (2017). · DOI · Paper page
  3. Majithia, A. R. et al. Prospective functional classification of all possible missense variants in PPARG. Nat Genet 48, 1570-1575, doi:10.1038/ng.3700 (2016). · DOI · Paper page
  4. Sun, S. et al. A proactive genotype-to-patient-phenotype map for cystathionine beta-synthase. Genome Med 12, 13, doi:10.1186/s13073-020-0711-1 (2020). · DOI
  5. Findlay, G. M., Boyle, E. A., Hause, R. J., Klein, J. C. & Shendure, J. Saturation editing of genomic regions by multiplex homology-directed repair. Nature 513, 120-123, doi:10.1038/nature13695 (2014). · DOI · Paper page
  6. Findlay, G. M. et al. Accurate classification of BRCA1 variants with saturation genome editing. Nature 562, 217-222, doi:10.1038/s41586-018-0461-z (2018). · DOI · Paper page
  7. Meitlis, I. et al. Multiplexed Functional Assessment of Genetic Variants in CARD11. Am J Hum Genet 107, 1029-1043, doi:10.1016/j.ajhg.2020.10.015 (2020). · DOI
  8. Kweon, J. et al. A CRISPR-based base-editing screen for the functional assessment of BRCA1 variants. Oncogene 39, 30-35, doi:10.1038/s41388-019- 0968-2 (2020). · DOI
  9. Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149-157, doi:10.1038/s41586-019-1711-4 (2019). · DOI · Paper page
  10. Carette, J. E. et al. Haploid genetic screens in human cells identify host factors used by pathogens. Science 326, 1231-1235, doi:10.1126/science.1178955 (2009). · DOI
  11. Vanier, M. T. Niemann-Pick disease type C. Orphanet J Rare Dis 5, 16, doi:10.1186/1750-1172-5-16 (2010). · DOI · Paper page
  12. Wassif, C. A. et al. High incidence of unrecognized visceral/neurological late- onset Niemann-Pick disease, type C1, predicted by analysis of massively parallel sequencing data sets. Genet Med 18, 41-48, doi:10.1038/gim.2015.25 (2016). · DOI
  13. Lin, Y. C. et al. Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations. Nat Commun 5, 4767, doi:10.1038/ncomms5767 (2014). · DOI
  14. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823, doi:10.1126/science.1231143 (2013). · DOI · Paper page
  15. Duan, J. et al. Genome-wide identification of CRISPR/Cas9 off-targets in human genome. Cell Res 24, 1009-1012, doi:10.1038/cr.2014.87 (2014). · DOI · Paper page
  16. Rose, J. C. et al. Suppression of unwanted CRISPR-Cas9 editing by co- administration of catalytically inactivating truncated guide RNAs. Nat Commun 11, 2697, doi:10.1038/s41467-020-16542-9 (2020). · DOI
  17. Coelho, M. A. et al. CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs. Nat Commun 11, 4132, doi:10.1038/s41467-020-17952-5 (2020). · DOI
  18. Kopitz, J., Gerhard, C., Hofler, P. & Cantz, M. [14C]Methylamine accumulation in cultured human skin fibroblasts--a biochemical test for lysosomal storage and lysosomal diseases. Clin Chim Acta 227, 121-133, doi:10.1016/0009- 8981(94)90141-4 (1994). · DOI
  19. Lachmann, R. H. et al. Treatment with miglustat reverses the lipid-trafficking defect in Niemann-Pick disease type C. Neurobiol Dis 16, 654-658, doi:10.1016/j.nbd.2004.05.002 (2004). · DOI
  20. te Vruchte, D. et al. Relative acidic compartment volume as a lysosomal storage disorder-associated biomarker. J Clin Invest 124, 1320-1328, doi:10.1172/JCI72835 (2014). · DOI
  21. Xu, M. et al. A phenotypic compound screening assay for lysosomal storage diseases. J Biomol Screen 19, 168-175, doi:10.1177/1087057113501197 (2014). · DOI
  22. Millat, G. et al. Niemann-Pick C1 disease: the I1061T substitution is a frequent mutant allele in patients of Western European descent and correlates with a classic juvenile phenotype. Am J Hum Genet 65, 1321-1329, doi:10.1086/302626 (1999). · DOI
  23. Millat, G. et al. Niemann-Pick C1 disease: correlations between NPC1 mutations, levels of NPC1 protein, and phenotypes emphasize the functional significance of the putative sterol-sensing domain and of the cysteine-rich luminal loop. Am J Hum Genet 68, 1373-1385, doi:10.1086/320606 (2001). · DOI
  24. Dardis, A. et al. Molecular Genetics of Niemann-Pick Type C Disease in Italy: An Update on 105 Patients and Description of 18 NPC1 Novel Variants. J Clin Med 9, doi:10.3390/jcm9030679 (2020). · DOI
  25. Millat, G. et al. Niemann-Pick C disease: use of denaturing high performance liquid chromatography for the detection of NPC1 and NPC2 genetic variations and impact on management of patients and families. Mol Genet Metab 86, 220- 232, doi:10.1016/j.ymgme.2005.07.007 (2005). · DOI
  26. Greer, W. L. et al. Mutations in NPC1 highlight a conserved NPC1-specific cysteine-rich domain. Am J Hum Genet 65, 1252-1260, doi:10.1086/302620 (1999). · DOI
  27. Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat Biotechnol 38, 824-844, doi:10.1038/s41587-020-0561-9 (2020). · DOI · Paper page
  28. Kim, H. K. et al. Predicting the efficiency of prime editing guide RNAs in human cells. Nat Biotechnol 39, 198-206, doi:10.1038/s41587-020-0677-y (2021). · DOI
  29. Rentzsch, P., Witten, D., Cooper, G. M., Shendure, J. & Kircher, M. CADD: predicting the deleteriousness of variants throughout the human genome. Nucleic Acids Res 47, D886-D894, doi:10.1093/nar/gky1016 (2019). · DOI · Paper page
  30. Gelsthorpe, M. E. et al. Niemann-Pick type C1 I1061T mutant encodes a functional protein that is selected for endoplasmic reticulum-associated degradation due to protein misfolding. J Biol Chem 283, 8229-8236, doi:10.1074/jbc.M708735200 (2008). · DOI
  31. Zampieri, S., Bembi, B., Rosso, N., Filocamo, M. & Dardis, A. Treatment of Human Fibroblasts Carrying NPC1 Missense Mutations with MG132 Leads to an Improvement of Intracellular Cholesterol Trafficking. JIMD Rep 2, 59-69, doi:10.1007/8904_2011_49 (2012). · DOI
  32. Watari, H. et al. Mutations in the leucine zipper motif and sterol-sensing domain inactivate the Niemann-Pick C1 glycoprotein. J Biol Chem 274, 21861-21866, doi:10.1074/jbc.274.31.21861 (1999). · DOI
  33. Kuchenbaecker, K. B. et al. Risks of Breast, Ovarian, and Contralateral Breast Cancer for BRCA1 and BRCA2 Mutation Carriers. JAMA 317, 2402-2416, doi:10.1001/jama.2017.7112 (2017). · DOI · Paper page
  34. Olopade, O. I. & Artioli, G. Efficacy of risk-reducing salpingo-oophorectomy in women with BRCA-1 and BRCA-2 mutations. Breast J 10 Suppl 1, S5-9, doi:10.1111/j.1524-4741.2004.101s3.x (2004). · DOI
  35. Rebbeck, T. R. et al. Bilateral prophylactic mastectomy reduces breast cancer risk in BRCA1 and BRCA2 mutation carriers: the PROSE Study Group. J Clin Oncol 22, 1055-1062, doi:10.1200/JCO.2004.04.188 (2004). · DOI · Paper page
  36. Hart, S. N. et al. Comprehensive annotation of BRCA1 and BRCA2 missense variants by functionally validated sequence-based computational prediction models. Genet Med 21, 71-80, doi:10.1038/s41436-018-0018-4 (2019). · DOI
  37. Kweon, J. et al. Engineered prime editors with PAM flexibility. Mol Ther, doi:10.1016/j.ymthe.2021.02.022 (2021). · DOI
  38. Liu, P. et al. Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice. Nat Commun 12, 2121, doi:10.1038/s41467-021- 22295-w (2021). · DOI
  39. Hua, K., Jiang, Y., Tao, X. & Zhu, J. K. Precision genome engineering in rice using prime editing system. Plant Biotechnol J 18, 2167-2169, doi:10.1111/pbi.13395 (2020). · DOI
  40. Erwood, S. et al. Modeling Niemann-Pick disease type C in a human haploid cell line allows for patient variant characterization and clinical interpretation. Genome Res 29, 2010-2019, doi:10.1101/gr.250720.119 (2019). · DOI
  41. Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol 36, 843-846, doi:10.1038/nbt.4172 (2018). · DOI · Paper page
  42. Labun, K. et al. CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res 47, W171-W174, doi:10.1093/nar/gkz365 (2019). · DOI
  43. Kluesner, M. G. et al. EditR: A Method to Quantify Base Editing from Sanger Sequencing. CRISPR J 1, 239-250, doi:10.1089/crispr.2018.0014 (2018). · DOI · Paper page
  44. Robinson, J. T. et al. Integrative genomics viewer. Nat Biotechnol 29, 24-26, doi:10.1038/nbt.1754 (2011). · DOI · Paper page
  45. Comaniciu, D. & Meer, P. Mean shift: a robust approach toward feature space analysis. IEEE Transactions on Pattern Analysis and Machine Intelligence 24, 603-619, doi:10.1109/34.1000236 (2002). · DOI

Cited by

Sorted by citations

Authors
Erwood, S., Bily, T. M. I., Lequyer, J., Yan, J., Gulati, N., Brewer, R. A., Zhou, L., Pelletier, L., Ivakine, E. A., & Cohn, R. D.
Affiliations
  • University of Toronto (CA)
  • Hospital for Sick Children (CA)
  • Mount Sinai Hospital (CA)
Corresponding author
Ivakine, Evgueni A.
ORCID
Venue
Nature Biotechnology
Publisher
Springer Science and Business Media LLC
Published
2022-02-21
Volume
40
Issue
6
Pages
885-895
ISSN
  • 0733-222X (ISSN-L)
  • 1087-0156
  • 1546-1696
DOI
10.1038/s41587-021-01201-1
OpenAlex
W4226280654
Cited by
163
References
8
Citations by year
2026: 20 · 2025: 29 · 2024: 32 · 2023: 54 · 2022: 23 · 2021: 5
FWCI
11.65
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Biotechnology (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
Funders
University of Pennsylvania Orphan Disease Center in partnership with The Andrew Coppola Foundation
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
  • Health Sciences › Medicine › Physiology › Lysosomal Storage Disorders Research
Keywords
variant interpretation, haploidization, NPC1, Niemann-Pick disease type C, missense mutations, deleterious mutation prediction, BRCA2, lysosomal storage disease
MeSH terms
Niemann-Pick C1 Protein, Humans, Mutation, Intracellular Signaling Peptides and Proteins, Niemann-Pick Disease, Type C, Clustered Regularly Interspaced Short Palindromic Repeats

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