Visible traits demonstrate that crispant founder mice can be used for phenotypic assessment

Reviewer #1 (Public review):

Summary:

This study evaluates the feasibility of using crispant founder mice, first-generation animals directly edited by CRISPR/Cas9, for initial phenotypic assessments. The authors target seven genes known to produce visible recessive traits to test whether mosaicism in founder animals prevents meaningful phenotype-genotype interpretation. Remarkably, they observe clear null phenotypes in founders for six of the seven genes, with high editing efficiencies. These results demonstrate that crispant mice can, under specific conditions, display recessive phenotypes that are readily interpretable. However, this conclusion should be moderated, as the study addresses only one biological context, visible Mendelian traits, and may not generalize to quantitative, subtle, or late-onset phenotypes. The report also examines attempts at multiplex CRISPR targeting, which reduce viability, underscoring limits for concurrent gene disruptions. Finally, the detailed description of diverse alleles generated by CRISPR provides valuable insight into how allelic series can be exploited to investigate protein function.

Strengths:

(1) The manuscript provides a comprehensive and technically rigorous description of CRISPR/Cas9‑induced mutations across several loci. The accompanying genotyping, sequencing, and analytical approaches are sound, complementary, and well-detailed, providing a resource that will be valuable to researchers using genome editing beyond the specific application of genetic screening.

(2) By documenting a wide diversity of alleles and mutation types, the study contributes to understanding how allelic series generated by CRISPR can be leveraged for dissecting protein function, a perspective that has been less systematically presented in prior literature and could be compared to targeted strategies such as those described by Cassidy et al. (2022, DOI: [10.1016/bs.mie.2022.03.053]) or other relevant studies addressing CRISPR-based allelic series generation.

(3) The work demonstrates technically solid editing and validation workflows, setting a methodological reference point for similar projects across species or trait categories.

Weaknesses:

(1) There is a disconnect between the abstract/introduction and the discussion. While both the abstract and introduction focus on the potential use of crispant founders for phenotypic assessment in the context of genetic screening, with the introduction notably emphasizing this framework through a detailed section on ENU-based screens, the discussion devotes relatively little attention to this aspect. Instead, it primarily examines CRISPR mutagenesis outcomes, mutation detection, and allele characterization. Overall, the study's aims are not clearly or explicitly defined, which contributes to the lack of alignment across sections.

(2) Important limitations of the approach are not sufficiently discussed. For instance, the paper does not address how applicable the findings are beyond visible Mendelian traits, such as for quantitative, late-onset, or more subtle phenotypes, including behavioral ones, or how to interpret wild-type appearing founders. There is little consideration of appropriate experimental controls (e.g., wild-type or mock-edited animals) or of how many animals might be required to robustly establish genotype-phenotype associations.

(3) The conclusion that this strategy will "dramatically reduce time, resources, and animal numbers" is not quantitatively supported by the data presented and should be expressed more cautiously.

Reviewer #2 (Public review):

Summary:

The authors sought to validate the use of genetic screening pipelines that assess phenotypes in founders (F0, referred to as "crispants") obtained from CRISPR/Cas9 gene editing in 1-cell zygotes. The application of this approach in mice has generally been avoided due to concerns that results would be confounded by genetic mosaicism, but benefits to this approach include reducing animal numbers needed to achieve goals of identifying knockout phenotypes, as well as improved efficiency in the use of time and resources. The authors targeted seven genes associated with visible recessive phenotypes and observed the expected null phenotype in up to 100% of founders for each gene. Although mosaicism was common in the crispants, the various alleles were generally all functional null alleles and, in fact, some in-frame deletions with null phenotypes revealed critical functional motifs within the gene products. The rigorous data presented support using crispants to assess knockout phenotypes when guide RNAs with strong on-target and low off-target scores are used for gene editing in 1-cell mouse embryos.

Strengths:

By targeting multiple genes with existing, well-characterized mutations, the authors established a robust system for validating the analysis of crispants to assess gene function.

Cutting-edge technologies were used to carefully assess the spectrum of mutations generated.

Weaknesses:

There could have been some discussion regarding how this approach would be impacted if mutations are dominant or embryonic lethal (for the latter, for example, F0 can be examined as embryos).

Reviewer #3 (Public review):

Summary:

The study assesses whether CRISPR-generated founder (F0) "crispant" mice can be reliably used for initial phenotypic assessment and screening. By targeting seven genes with known visible recessive phenotypes, the authors show that, despite genetic mosaicism, the expected null phenotypes were observed in all targeted genes. These findings demonstrate that the phenotyping and screening of F0 "crispant" mice is a valid (and efficient) approach to selecting candidate alleles for follow-up studies, thereby streamlining mouse breeding and animal husbandry-related costs.

Strengths:

The study is comprehensive, carefully executed, and provides deep insight into the utility of F0 "crispant" mice for phenotypic screening. The authors evaluated the CRISPR/Cas9 editing outcomes across seven genes using multiple sequencing modalities, providing a robust framework for determining and interpreting complex founder genotypes. Importantly, the study examines/highlights the biological insight gained from compound heterozygous founders and naturally arising allelic series, enabling genotype-phenotype associations and functional inferences about protein domains.

More broadly, the authors' thorough evaluation of the CRISPR/Cas9-based gene editing events in the founders can serve as a benchmark for others in the field, engineering their own mouse "crispants."

Weaknesses:

The relationship between the sgRNA/Cas9 concentrations delivered to the zygotes and the resulting editing efficiencies are not explicitly investigated.

Author response:

We would like to thank the reviewers for their detailed reading of our manuscript and for the constructive comments they have provided.

We plan to make structural changes to the introduction and the discussion. Reviewer #1 describes the “disconnect between the abstract/introduction and the discussion”. We agree that “the study's aims are not clearly or explicitly defined”. We will edit the introduction to state our aim of investigating the factors that affect using “crispants” in mouse functional genomics. In the discussion, we described how our findings inform sgRNA choice to ensure biallelic gene disruption in founders and how our extensive genotyping methods enabled us to determine the molecular basis for the observed phenotype (explaining why some founders showed the expected recessive trait and why it was partial or absent in others). We also concluded from our attempts of multiplexing that this had too great an impact on viability to be useful. We will edit the discussion to better address our aim and to elaborate on several points raised by the reviewers (discussed in more detail below). Specifically, we will provide examples of screening situations where generating crispant mice may be useful, e.g. preliminary in vivo studies to follow up candidates identified in large-scale cellular screens. We will also provide more context about our assumptions underlying our statement that the use of crispants will “dramatically reduce time, resources, and animal numbers” compared to ENU mutagenesis (where recessive traits require breeding of G2 females with G1 males to achieve homozygosity of de novo mutations in G3 offspring) and the work needed to validate this. We will more clearly acknowledge that our proof-of-principle study used visible phenotypes that can be assessed in individual animals and then discuss how the use of crispants could be extended to the investigation of quantitative or late-onset traits using cohorts of crispants (discussed further below). We will also discuss the assessment of non-null alleles to dissect protein function, building on our unexpected finding that a single round of CRISPR/Cas9mediated mutagenesis can generate an allelic series.

Reviewer #1 asked us to address “how to interpret wild-type appearing founders”. We have discussed the mechanisms underlying the wild-type appearing founders generated in this study. This is linked with concerns in the field that incomplete editing, transcripts escaping nonsense-mediated decay, and/or the presence of in-frame mutations that don’t disrupt protein function may lead to founders that appear wild-type or have a partial phenotype. We have shown that our electroporation protocol results in very high levels of editing, but that this must always be assessed during genotyping. We found that by using an sgRNA that targets a critical protein domain, you can ensure that short in-frame indels also disrupt protein function. In future studies that determine how strain background modifies a phenotype that has been established on one strain (e.g. C57BL/6J), wild-type appearing founders would suggest that the new strain background rescues the null phenotype. In future studies that determine the consequence of targeting a second gene on a mutant background, wild-type appearing founders would indicate that the second mutation supresses the phenotype associated with the mutant background. We will add this to the discussion section where we describe possible screening situations in which crispant mice would be useful.

Reviewer #3 states that “the relationship between the sgRNA/Cas9 concentrations delivered to the zygotes and the resulting editing efficiencies are not explicitly investigated.” Members of The Centre for Phenogenomics (TCP) Transgenic Production Core who co-author this study (Lauryl Nutter, Marina Gertsenstein and Lauri Lintott) have published detailed protocols on mouse model production, which we cite in this paper (PMID: 30040228; PMID: 33524495; PMID: 39999224). In PMID: 33524495, they tested a two-fold difference in Cas9 RNP concentrations for generating knock-out alleles. Using their optimised protocols for electroporation of one cell zygotes with RNPs, we achieved an extremely high editing rate. We did not vary the sgRNA/Cas9 concentrations as part of this study as our goal was to assess the ability to generate “complete” null animals. We do note, however, that by targeting two genes simultaneously whilst keeping the total RNP concentration constant (to avoid reagent toxicity), we halved the amount of each sgRNA and this did not lead to a decrease in editing efficiency. We will highlight this in the results/discussion section (as appropriate).

Reviewer #1 asks about whether the use of crispants is applicable for “quantitative, late-onset, or more subtle phenotypes, including behavioral ones”. We are hopeful that this is possible and it is a priority for future studies. Crucially, cohorts of crispants can be generated in a single round of mutagenesis. Starting an experiment with ten donor females will produce ~100 zygotes, resulting in ~40 crispants. Power calculations must be performed to determine the size of the cohort required for the effect size and variability of the phenotype being studied, but many neurobehavioural studies use ~10 mutants vs ~10 controls. We note that sex and/or background genotype may mean that only some of the ~40 crispants produced can be used for phenotypic testing. This reviewer also raises the point about whether wild-type animals or mock-edited animals serve as the best controls. From work carried out by Lauryl Nutter and her colleagues from the IMPC (PMID: 37301944), we know that “wild-type” controls should ideally be from the same embryo pool as the crispants to avoid differences due to genetic drift within inbred colonies. This study also found that possible off-target mutations from CRISPR/Cas9-mediated mutagenesis is not an issue (despite a lot of attention in the literature). The suggestion of using mock-edited controls, resulting from zygotes that have gone through electroporation without RNP, addresses a possible need to control for the stress of undergoing the electroporation process. Our study shows that additional stress is caused by inducing and repairing a break in a neutral locus (EGFP). Controlling for these stressors may be particularly important when assessing behavioural phenotypes in crispants vs controls.

Reviewer #2 states that “there could have been some discussion regarding how this approach would be impacted if mutations are dominant or embryonic lethal (for the latter, for example, F0 can be examined as embryos).” Our manuscript discusses how crispants could help with the study of genes that may be essential. Specifically, we stated that when CRISPR/Cas9-mediated mutagenesis fails to produce live pups, phenotypic assessment of crispant embryos could reveal whether targeting the gene impacts embryogenesis. Crispants can only be used to screen for recessive traits since both alleles are edited. The assessment of dominant traits is not addressed in our study and remains a challenge in the field. We note that CRISPRi screens in cultured cells reveal candidates that when partially downregulated lead to the desired phenotype. One possibility is to employ this set up in vivo using dCas9-KRAB transgenic mice (JAX stock #030000). We could add this point to the discussion section.