Cergentis news

Transgenic mouse model

Cas9-induced double-stranded break in a gene regulatory region triggers unexpected gene activation

| |
5 min read

The advent of CRISPR/Cas9 genome editing tool has revolutionized biomedical research.1-6 Its added-value is also apparent in research focusing on DNA damage response.7-11 Since the introduction of these game-changing “molecular scissors”, several systems have been explored to deliver CRISPR/Cas9 components into the nucleus, to promote efficient genome editing. Among the delivery strategies, we count lentiviral vectors.6,12,13

Given that little is known about the impact of viral CRISPR/Cas9 delivery methods on genome integrity and gene expression - following random integration of the construct into the host genome14 - scientists at the Netherlands Cancer Institute (NKI) and those affiliated with Oncode Institute sought to elucidate how lentiviral-based sgRNA vector affects the expression of a target gene. Their findings were published in EMBO reports.15

Lentiviral vector integrates upon a DSB in the ABCB1 promoter and drives gene expression

The authors previously found that human RPE-1 (retinal pigment ephitilial-1) can acquire Taxol resistance through transcriptional activation of ABCB1 gene. In this study, they leveraged LentiGuide-Puro and cloned different sgRNAs targeting different noncoding regions across the ABCB1 locus to generate double-stranded break (DSB).15-17 Noncoding regions were purposely singled out to avoid the possibility that a break-induced change in coding sequence would lead to the acquisition of a Taxol-resistant phenotype. Interestingly, a subset of cells acquired Taxol-resistance via upregulation of ABCB1.15

To ensure that no genetic variations nor structural variants (e.g. translocations or insertions) might have accompanied their editing, the authors availed themselves of our proprietary genetic QC solutions.18 As such, RPE-1 parental cells were compared to the (sg6C9) Taxol-resistant clone derived from the sgRNA #6. Surprisingly, it was found that EEF1A1 promoter was amplified in the sg6C9 Taxol-resistant clone. In fact, our genome-wide (TLA) coverage suggested genomic insertions. To confirm the fusion of the ABCB1 and EEF1A1, the researchers carried out PCRs on genomic DNA. Results confirmed that the EEF1A1 promoter was integrated at the break site in the regulatory region of ABCB1. Additionally, sequencing the PCR products from various clones also revealed the presence of other sequences (i.e. U6 promoter and the puromycin-resistant cassette). Ultimately, the scientists were able to work out that the EEF1A1 integration found in the ABCB1 promoter belonged to the lentiviral vector and not to the endogenous gene found on chromosome 6.15

All in all, upon inducing a DSB in the regulatory region of ABCB1 gene, the researchers produced Taxol-resistant clones that upregulated ABCB1 through transcriptional activation via the EEF1A1 promoter from the vector.15

An unreported CRISPR/Cas9 on-target effect using sgRNA lentiviral method

In sum, the authors conclude that “a lentiviral sgRNA delivery system used to induce a DSB close to the transcriptional start site of a gene can result in the integration of the vector in the break site and subsequent activation of the gene.” For this reason, they suggest drawing on non-integrative systems instead, to investigate long-term effects of DNA damage response.15,19

The need to make genetic QC more common practice

The ability of our TLA-based approach to robustly detect all genetic variation (including structural variants) in and around genes of interest has captured the attention of many biomedical researchers globally. In fact, TLA-based assays are routinely adopted to help troubleshoot some of the most pressing genetic characterization challenges and our unique capabilities have been described in over 50 peer-reviewed scientific publications. On many occasions, we helped uncovered unexpected genetic events accompanying genetic engineering. We summarize below some notable published studies:

  • Genentech sought to map the exact location of integrated sequences in 7 previously published Cre and CreERT2 transgenic lines. Not only our proprietary solutions determined the number of integration sites as well as their exact positions in the genome, but we also identified unexpected structural changes accompanying those integrations.
  • In a collaborative study with The Jackson Laboratory, our solutions were applied to identify potential insertional mutagenesis. This was a large-scale study that aimed at analyzing transgene insertion sites from 40 highly used transgenic mouse lines. Here, TLA-based assays revealed the unexpected co-integration E.coli sequences, in 10 out of 40 animal models, that had co-integrated with the transgene sequence. As a result, these findings clearly highlight a need for more careful control strategies.
  • In a recent study conducted by a team at the Medical University of Innsbruck were confronted with a tandem duplication had occurred at the locus of homologous recombination during the generation of a novel Agmo KO mouse model. This unexpected structural event ended up fooling genotyping, as conventional assays such as FISH or long-range PCR were also unable to resolve the unexpected large structural variation. Therefore, these limitations underscore the shortcomings of classical routine genotyping strategies to profile structural variations in transgenic models.

For a complete overview of all peer-reviewed scientific publications describing the added-value of our proprietary TLA-based solutions, we invite you to consult our publications page, where you will be able to further filter by application of interest.





[1] Mojica FJM, Díez-Villaseñor C, García-Martínez J, Soria E (2005) Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol 60: 174–182

[2] van der Oost J, Jore MM, Westra ER, Lundgren M, Brouns SJJ (2009) CRISPR-based adaptive and heritable immunity in prokaryotes. Trends Biochem Sci 34: 401–407

[3] Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816–821

[4] Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339: 819–823

[5] Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339: 823–826

[6] Lino CA, Harper JC, Carney JP, Timlin JA (2018) Delivering crispr: a review of the challenges and approaches. Drug Deliv 25: 1234–1257

[7] Vítor AC, Huertas P, Legube G, de Almeida SF (2020) Studying DNA double-strand break repair: an ever-growing toolbox. Front Mol Biosci 7: 24

[8] Aymard F, Aguirrebengoa M, Guillou E, Javierre BM, Bugler B, Arnould C, Rocher V, Iacovoni JS, Biernacka A, Skrzypczak M et al (2017) Genome-wide mapping of long-range contacts unveils clustering of DNA double-strand breaks at damaged active genes. Nat Struct Mol Biol 24: 353–361

[9] D’Alessandro G, d’Adda di Fagagna F (2017) Transcription and DNA damage: holding hands or crossing swords? J Mol Biol 429: 3215–3229

[10] Clouaire T, Legube cuG (2019) A snapshot on the cis chromatin response to DNA double-strand breaks. Trends Genet 35: 330–345

[11] Miné-Hattab J, Chiolo I (2020) Complex chromatin motions for DNA repair. Front Genet 11: 800

[12] Warnock JN, Daigre C, Al-Rubeai M (2011) Introduction to viral vectors. Methods Mol Biol 737: 1–25

[13] Mulero-Sánchez A, Pogacar Z, Vecchione L (2019) Importance of genetic screens in precision oncology. ESMO Open 4: e000505

[14] Kotterman MA, Chalberg TW, Schaffer DV (2015) Viral vectors for gene therapy: translational and clinical outlook. Annu Rev Biomed Eng 17: 63–89

[16] Tame MA, Manjón AG, Belokhvostova D, Raaijmakers JA, Medema RH (2017) TUBB3 overexpression has a negligible effect on the sensitivity to taxol in cultured cell lines. Oncotarget 8: 71536

[17] Manjón AG, Hupkes DP, Liu NQ, Friskes A, Joosten S, Teunissen H, Aarts M, Prekovic S, Zwart W, De Wit E et al (2021) Perturbations in 3D genome organization can promote acquired drug resistance. bioRxiv https://doi.org/10.1101/2021.02.02.429315 [PREPRINT]

[18] de Vree PJP, de Wit E, Yilmaz M, van de Heijning M, Klous P, Verstegen MJAM, Wan YI, Teunissen H, Krijger PHL, Geeven G et al (2014) Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Nat Biotechnol 32: 1019–1025

[19] Yan S, Schubert M, Young M, Wang B (2017) Applications of Cas9 nickases for genome engineering

© 2012-2022 Cergentis B.V. All rights reserved.
For Research Use Only. Not for use in diagnostic procedures.