Simple and reliable detection of CRISPR-induced on-target effects by qgPCR and SNP genotyping

Isabel Weisheit, Joseph A. Kroeger, Rainer Malik, Benedikt Wefers, Peter Lichtner, Wolfgang Wurst, Martin Dichgans, Dominik Paquet

Published: 2021-02-16 DOI: 10.1038/s41596-020-00481-2

CRISPR-induced OnTEs

qgPCR

SNP genotyping

genome editing

allele detection

AI 解读

Supplementary information

Supplementary Table 1

Primer, sgRNA and ssODN sequences

Author Correction: Full-length sequencing of circular DNA viruses and extrachromosomal circular DNA using CIDER-Seq

Generation of heart-forming organoids from human pluripotent stem cells

Design and fabrication of wearable electronic textiles using twisted fiber-based threads

Activation of mechanoluminescent nanotransducers by focused ultrasound enables light delivery to deep-seated tissue in vivo

Chromatin accessibility profiling by ATAC-seq

Deoxyfluorination of phenols for chemoselective18F-labeling of peptides

Author Correction: cfSNV: a software tool for the sensitive detection of somatic mutations from cell-free DNA

Human primordial germ cell-like cells specified from resetting precursors develop in human hindgut organoids

An inexpensive semi-automated sample processing pipeline for cell-free RNA extraction

Application of CHyMErA Cas9-Cas12a combinatorial genome-editing platform for genetic interaction mapping and gene fragment deletion screening

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References

Supplementary information

Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).
Wright, A. V., Nuñez, J. K. & Doudna, J. A. Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell 164, 29–44 (2016).
Komor, A. C., Badran, A. H. & Liu, D. R. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell 168, 20–36 (2017).
Fellmann, C., Gowen, B. G., Lin, P.-C., Doudna, J. A. & Corn, J. E. Cornerstones of CRISPR-Cas in drug discovery and therapy. Nat. Rev. Drug Discov. 16, 89–100 (2017).
Clement, K., Hsu, J. Y., Canver, M. C., Joung, J. K. & Pinello, L. Technologies and computational analysis strategies for CRISPR applications. Mol Cell 79, 11–29 (2020).
Pickar-Oliver, A. & Gersbach, C. A. The next generation of CRISPR-Cas technologies and applications. Nat. Rev. Mol. Cell Biol. 20, 1–18 (2019).
Wienert, B., Wyman, S. K., Yeh, C. D., Conklin, B. R. & Corn, J. E. CRISPR off-target detection with DISCOVER-seq. Nat. Protoc. 15, 1–28 (2020).
Gkazi, S. A. Quantifying CRISPR off-target effects. Emerg. Top. Life Sci. 3, 327–334 (2019).
Shin, H. Y. et al. CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome. Nat. Commun. 8, 15464 (2017).
Adikusuma, F. et al. Large deletions induced by Cas9 cleavage. Nature 560, E8–E9 (2018).
Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36, 765–771 (2018).
Owens, D. D. G. et al. Microhomologies are prevalent at Cas9-induced larger deletions. Nucleic Acids Res. 47, 7402–7417 (2019).
Weisheit, I. et al. Detection of deleterious on-target effects after HDR-mediated CRISPR editing. Cell Rep. 31, 107689 (2020).
Ikeda, K. et al. Efficient scarless genome editing in human pluripotent stem cells. Nat. Methods 15, 1–7 (2018).
Bustin, S. A. & Nolan, T. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J. Biomol. Tech. 15, 155–166 (2004).
Nolan, T., Hands, R. E. & Bustin, S. A. Quantification of mRNA using real-time RT-PCR. Nat. Protoc. 1, 1559–1582 (2006).
Bi, C. et al. Long-read individual-molecule sequencing reveals CRISPR-induced genetic heterogeneity in human ESCs. Genome Biol. 21, 213 (2020).
Ho, S. S., Urban, A. E. & Mills, R. E. Structural variation in the sequencing era. Nat. Rev. Genet. 21, 171–189 (2019).
Kosugi, S. et al. Comprehensive evaluation of structural variation detection algorithms for whole genome sequencing. Genome Biol. 20, 117 (2019).
Cullot, G. et al. CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations. Nat. Commun. 10, 1–14 (2019).
Yates, A. D. et al. Ensembl 2020. Nucleic Acids Res. 41, D21–D27 (2019).
Illumina. Infinium Global Screening Array-24 v3.0 BeadChip 1–6 (accessed 22 July 2020); https://www.illumina.com/products/by-type/microarray-kits/infinium-global-screening.html
Hashimoto, M., Yamashita, Y. & Takemoto, T. Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Dev. Biol. 418, 1–9 (2016).
Tu, Z. et al. Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos. Sci. Rep. 7, 1–11 (2017).
Li, Y. et al. Precise allele-specific genome editing by spatiotemporal control of CRISPR-Cas9 via pronuclear transplantation. Nat. Commun. 11, 4593 (2020).
Bogue, M. A., Churchill, G. A. & Chesler, E. J. Collaborative cross and diversity outbred data resources in the mouse phenome database. Mamm. Genome 26, 511–520 (2015).
Kwart, D., Paquet, D., Teo, S. & Tessier-Lavigne, M. Precise and efficient scarless genome editing in stem cells using CORRECT. Nat. Protoc. 12, 329–354 (2017).
Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 (2016).
Wefers, B., Bashir, S., Rossius, J., Wurst, W. & Kühn, R. Gene editing in mouse zygotes using the CRISPR/Cas9 system. Methods 121-122, 55–67 (2017).
Modzelewski, A. J. et al. Efficient mouse genome engineering by CRISPR-EZ technology. Nat. Protoc. 13, 1253–1274 (2018).
Hruscha, A. et al. Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish. Development 140, 4982–4987 (2013).
Taylor, S. C. et al. The ultimate qPCR experiment: producing publication quality, reproducible data the first time. Trends Biotechnol. 37, 761–774 (2019).
Weissenborn, S. J., Wieland, U., Junk, M. & Pfister, H. Quantification of beta-human papillomavirus DNA by real-time PCR. Nat. Protoc. 5, 1–13 (2010).
Haass, C. et al. The Swedish mutation causes early-onset Alzheimer’s disease by beta-secretase cleavage within the secretory pathway. Nat. Med. 1, 1291–1296 (1995).
Sullivan, S. et al. Quality control guidelines for clinical-grade human induced pluripotent stem cell lines. Regen. Med. 13, 859–866 (2018).
Baker, D. et al. Detecting genetic mosaicism in cultures of human pluripotent stem cells. Stem Cell Rep. 7, 998–1012 (2016).
Concordet, J.-P. & Haeussler, M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 46, W242–W245 (2018).
Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).
Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191 (2016).
Tycko, J. et al. Mitigation of off-target toxicity in CRISPR-Cas9 screens for essential non-coding elements. Nat. Commun. 10, 4063 (2019).
Duncan, G. T., Balamurugan, K., Budowle, B., Smerick, J. & Tracey, M. L. Microvariation at the human D1S80 locus. Int. J. Legal Med. 110, 150–154 (1997).
Zurita, E. et al. Genetic polymorphisms among C57BL/6 mouse inbred strains. Transgenic Res. 20, 481–489 (2011).
Mattapallil, M. J. et al. The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes. Invest. Ophthalmol. Vis. Sci. 53, 2921–2927 (2012).
Freeman, H. C., Hugill, A., Dear, N. T., Ashcroft, F. M. & Cox, R. D. Deletion of nicotinamide nucleotide transhydrogenase: a new quantitive trait locus accounting for glucose intolerance in C57BL/6J mice. Diabetes 55, 2153–2156 (2006).
Bouma, M. J. et al. Differentiation-defective human induced pluripotent stem cells reveal strengths and limitations of the teratoma assay and in vitro pluripotency assays. Stem Cell Rep. 8, 1340–1353 (2017).

Simple and reliable detection of CRISPR-induced on-target effects by qgPCR and SNP genotyping

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