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Unmasking Challenges of CRISPR in Mouse Model Research

Unmasking Challenges of CRISPR in Mouse Model Research

Unmasking Challenges of CRISPR in Mouse Model Research

December 12, 2023
CRISPR in Mouse Model Research

CSRIPR-cas9 genome editing promises an exciting and diverse array of research opportunities from personalised medicine to agriculture due to its ability to generate targeted genomic alterations in a diverse range of cell lines and species. This includes animal models that play a key role in the study of physiology, disease aetiology and progression, development of therapeutics, and pre-clinical trials. In the context of the continuing evolution and maturation of this technology there is understandable excitement regarding the potential of genomic engineering. In such an environment, it is important not to overlook the limitations and potential pitfalls that are inherent to the technology when deciding if CRISPR-Cas9 is the best tool to apply to attaining your research goals and objectives.

The publication exclusively of successful research can give researchers the impression that genome engineering is always simple, cheap, and efficient. A recent survey of actual bench-top experience of over 200 researchers from a diverse range of roles, utilising CRISPR in a diverse array of applications, found that genome engineering approaches are not always simple or effective1. Researchers reported that the duration of a typical CRISPR workflow was 10 weeks. However, it was necessary to repeat a CRISPR experiment an average of seven times due to factors such as sub-optimal editing efficiency, before obtaining the desired genetic modification. This means that the true timeline required to obtain successfully edited lines typically approximates 70 weeks. This additional time and re-work also blew out associated cost to in excess of $18000 on average. Almost half of researchers surveyed did not analyse what indels were generated by their editing, which is particularly surprising given that editing efficiencies can be low, often resulting in no editing at all, or can result in a wide array of genetic alterations, many of which may be far from desirable.

“This means that the true timeline required to obtain successfully edited lines typically approximates 70 weeks”

CRISPR induced DNA lesions such as the double strand breaks introduced by classical Cas9 nucleases must be repaired by any of a number of cellular DNA repair pathways that include non-homologous end joining (NHEJ), microhomology mediated end joining (MMEJ) and homology directed repair (HDR)2. Whilst HDR repairs a break with fidelity, it occurs at a lower frequency than competing mutative repair pathways with the result that genetic alterations induced by CRISPR at the target site, known as on-target effects, can be diverse and not always desirable. Undesirable on-target effects are being reported in a growing body of literature3. These include very large deletions, chromosomal rearrangements and genomic instability4–10.

Whilst the use of nickases, that introduce a single strand break, and base editors, that chemically modify bases, can lower the incidence of on-target effects, undesirable outcomes still occur. The introduction of single strand breaks by nickases can result in the collapse of replication forks and the introduction of double strand breaks. One study found a similar prevalence of large deletions using either a Cas9 nuclease, or nickase in mouse embryos11. Whole genome sequencing of human induced pluripotent stem cells following base editor expression identified off-target point mutations beyond those intended and even beyond those predicted in silico12.

Even successful generation of on-target mutations, such as the introduction of indels for the generation of a knock-out line can have unforeseen consequences. While the expectation is that the presence of an indel in an exon will generate a premature stop codon, eliciting nonsense-mediated decay of the affected transcript, it has been shown that the presence of indels in transcripts can promote internal ribosome entry, convert alternatively spliced transcripts into protein encoding transcripts, and induce aberrant mRNA splicing13.

The creation of targeted knock-in and conditional knockout alleles by CRSIPR requires the introduction of a repair template into the genome by HDR. One factor limiting this approach is that HDR occurs at lower frequency than competing repair pathways, particularly in human cell lines, which is why much effort has been invested in approaches that favour the HDR pathway. Even so, the rates of efficiency of correct integration of the repair template and generation of the desired knock-in allele vary. Careful screening for the correct HDR event with the donor DNA is required as donor DNA templates can often integrate in a head-to-tail orientation14. The size of knock-in alleles is therefore limited in CRISPR-based approaches.

In the generation of genetically modified mouse lines, CRISPR components are often introduced directly into zygotes. The Cas9 nuclease can continue to be active through multiple rounds of cell division in the early embryo, although this effect may be reduced by the electroporation of Cas9 ribonucleoprotein into very early-stage zygotes. The high probability of CRISPR-induced DNA lesions being repaired by competing pathways often results in founder animals being mosaics of multiple different alleles15,16. This causes uncertainty as to the genetic composition of the germline of resulting founder lines.

It has long been recognised that Cas9 will cleave DNA at sites in the genome other than that being targeted17. Although these off-target effects can be reduced by using improved nucleases and improving guide RNA design18–20, it is still important to consider potential off-target effects in applications where they are particularly undesirable.

The possible generation of a diverse array of alleles in mosaic founder lines, the low efficiency of HDR for the generation of knock-in and conditional knockout lines, and the possible generation of undesirable and unexpected genetic alterations, both on and off-target, all mean that the outcome of a CRISPR-based approach in the generation of animal models is uncertain. Careful consideration of genotyping approaches and methodology is required for screening of founder mice to determine which, if any, contain the desired genetic modification and to ensure that the possible presence of large or complex on-target genetic alterations is not overlooked21–23. Breeding of founder lines is required to verify the desired edit is transmitted to the next generation and to segregate unwanted off-target edits.  This process can be time-consuming and result in the generation of a large number of animals.

In contrast to the uncertainty that can accompany the generation of animal lines following CRISPR-based approaches, particularly for large or complex alleles, goGermline™ technology offers an improved degree of certainty24. goGermline™ mice containing defined heterozygous genetic modification, generated in as little as 20 weeks, can be used to initiate the breeding of experimental cohorts of animals directly from the chimera stage without the need for extensive screening and genotyping of CRISPR-generated founder lines. In contrast to the limited size of CRISPR-knock-ins, OzBIG permits large knock-ins such as genomic humanizations of up to 240kb.

“goGermline™ mice containing defined heterozygous genetic modification, generated in as little as 20 weeks, can be used to initiate the breeding of experimental cohorts of animals directly from the chimera stage”

Whilst CRISPR-Cas9 genome editing techniques continue to diversify and refine across a broad array of applications, the potential pitfalls and uncertainty that may be encountered should be considered prior to embarking in the generation of any genetically modified mouse line.

CRISPR
  1. CRISPR Benchmark Report: A Genome Engineering Survey. Synthego https://www.synthego.com/crispr-benchmark.
  2. Nambiar, T. S., Baudrier, L., Billon, P. & Ciccia, A. CRISPR-based genome editing through the lens of DNA repair. Mol. Cell 82, 348–388 (2022).
  3. Boutin, J. et al. ON-Target Adverse Events of CRISPR-Cas9 Nuclease: More Chaotic than Expected. CRISPR J. 5, 19–30 (2022).
  4. Rayner, E. et al. CRISPR-Cas9 Causes Chromosomal Instability and Rearrangements in Cancer Cell Lines, Detectable by Cytogenetic Methods. CRISPR J. 2, 406–416 (2019).
  5. 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).
  6. Cullot, G. et al. CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations. Nat. Commun. 10, 1136 (2019).
  7. Rezza, A. et al. Unexpected genomic rearrangements at targeted loci associated with CRISPR/Cas9-mediated knock-in. Sci. Rep. 9, 3486 (2019).
  8. Simeonov, D. R. et al. A large CRISPR-induced bystander mutation causes immune dysregulation. Commun. Biol. 2, 70 (2019).
  9. Papathanasiou, S. et al. Whole chromosome loss and genomic instability in mouse embryos after CRISPR-Cas9 genome editing. Nat. Commun. 12, 5855 (2021).
  10. Leibowitz, M. L. et al. Chromothripsis as an on-target consequence of CRISPR–Cas9 genome editing. Nat. Genet. 53, 895–905 (2021).
  11. Owens, D. D. G. et al. Microhomologies are prevalent at Cas9-induced larger deletions. Nucleic Acids Res. 47, 7402–7417 (2019).
  12. McGrath, E. et al. Targeting specificity of APOBEC-based cytosine base editor in human iPSCs determined by whole genome sequencing. Nat. Commun. 10, 5353 (2019).
  13. Tuladhar, R. et al. CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation. Nat. Commun. 10, 4056 (2019).
  14. Skryabin, B. V. et al. Pervasive head-to-tail insertions of DNA templates mask desired CRISPR-Cas9–mediated genome editing events. Sci. Adv. 6, eaax2941 (2020).
  15. Yen, S.-T. et al. Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Dev. Biol. 393, 3–9 (2014).
  16. Mianné, J. et al. Analysing the outcome of CRISPR-aided genome editing in embryos: Screening, genotyping and quality control. Methods San Diego Calif 121–122, 68–76 (2017).
  17. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).
  18. Yu, Y. et al. Cytosine base editors with minimized unguided DNA and RNA off-target events and high on-target activity. Nat. Commun. 11, 2052 (2020).
  19. Jin, S. et al. Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity. Mol. Cell 79, 728-740.e6 (2020).
  20. Villiger, L. et al. In vivo cytidine base editing of hepatocytes without detectable off-target mutations in RNA and DNA. Nat. Biomed. Eng. 5, 179–189 (2021).
  21. Frequent loss of heterozygosity in CRISPR-Cas9–edited early human embryos. https://www.pnas.org/doi/10.1073/pnas.2004832117 doi:10.1073/pnas.2004832117.
  22. Hunt, J. M. T., Samson, C. A., Rand, A. du & Sheppard, H. M. Unintended CRISPR-Cas9 editing outcomes: a review of the detection and prevalence of structural variants generated by gene-editing in human cells. Hum. Genet. 142, 705–720 (2023).
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  24. Koentgen, F. et al. Exclusive transmission of the embryonic stem cell‐derived genome through the mouse germline. Genes. N. Y. N 2000 54, 326–333 (2016).

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