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OPEN Strategies to reduce genetic

mosaicism following CRISPR-
mediated genome edition in bovine
Received: 10 November 2018
Accepted: 30 September 2019 embryos
Published: xx xx xxxx
I. Lamas-Toranzo   , B.  Galiano-Cogolludo, F. Cornudella-Ardiaca, J. Cobos-Figueroa,
O. Ousinde & P. Bermejo-Álvarez   
Genetic mosaicism is the presence of more than two alleles on an individual and it is commonly
observed following CRISPR microinjection of zygotes. This phenomenon appears when DNA replication
precedes CRISPR-mediated genome edition and it is undesirable because it reduces greatly the odds
for direct KO generation by randomly generated indels. In this study, we have developed alternative
protocols to reduce mosaicism rates following CRISPR-mediated genome edition in bovine. In a
preliminary study we observed by EdU incorporation that DNA replication has already occurred at the
conventional microinjection time (20 hpi). Aiming to reduce mosaicism appearance, we have developed
three alternative microinjection protocols: early zygote microinjection (10 hpi RNA) or oocyte
microinjection before fertilization with either RNA or Ribonucleoprotein delivery (0 hpi RNA or 0 hpi
RNP). All three alternative microinjection protocols resulted in similar blastocyst and genome edition
rates compared to the conventional 20 hpi group, whereas mosaicism rates were significantly reduced
in all early delivery groups (~10–30% of edited embryos being mosaic depending on the loci) compared
to conventional 20 hpi microinjection (100% mosaicism rate). These strategies constitute an efficient
way to reduce the number of indels, increasing the odds for direct KO generation.

Genome modification at specific loci allows the ablation (knock-out, KO) or insertion (knock-in, KI) of specific
DNA sequences to unequivocally assess the role of a specific gene on a particular physiological process or to alter
the phenotype of an animal for diverse purposes. Unfortunately, technical limitations have largely restricted targeted
genome modification in mammals to the mouse model. These limitations derived from the extremely low efficiency
of the only available technique for targeted mutagenesis, homologous recombination (HR)1, which impedes its direct
application on embryos. The low efficiencies of HR could only be bypassed by the use of embryonic stem cells (ESC)
as an intermediary to generate targeted genetically modified mice2. Later, the development of somatic cell nuclear
transfer (SCNT) allowed the use of somatic cells as intermediaries for HR to generate genetically modified animals
in other species where truly pluripotent ESC were not available3, but the complexity and inefficiency of SCNT has
impeded its widespread use. The advent of site-specific endonucleases has situated genome modification at an attain-
able distance in these species, empowering scientist to perform loss-of-function experiments or to generate geneti-
cally modified animals for different applications4. Among the different site-specific endonucleases, CRISPR, the last
technology to be developed has become the method of choice given its ease of use and flexibility5.
CRISPR technology allows KO generation in a single-step by microinjection at the zygote stage6. For this purpose,
CRISPR is directed to the beginning of the coding region of the target gene, where it will induce a double-strand
break (DSB). The DSB can be repaired by either HR, which reconstitutes the target site allowing CRISPR recogni-
tion and thereby the generation of another DSB, or by non-homologous end joining repair (NHEJ), which often
generates random insertions or deletions (indels) at the target site. Those indels constitute a stable mutation, as they
impede target recognition by CRISPR, and can produce KO alleles, as those indels not multiple of three disrupt the
open reading frame (ORF) of the target gene, leading to a truncated protein. However, KO generation in one step
(i.e. one pregnancy) requires all alleles harboured by a given individual to be KO (i.e., not multiple of three) and,
therefore, a reduction in the number of alleles generated greatly increases the odds for direct KO generation.

Animal Reproduction Department, INIA, Madrid, Spain. Correspondence and requests for materials should be
addressed to P.B.-Á. (email: [email protected])

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No. of embryos genotyped No. of embryos No. of embryos genotyped No. of mosaic
Group by PCR sequencing edited (%) by clonal sequencing embryos (%)
RNP-injected 0 hpi 23 20 (87.0) 20 6 (30.0)a
mRNA-injected 0 hpi 25 22 (88.0) 20 6 (30.0)a
mRNA-injected 10 hpi 24 20 (83.3) 20 7 (35.0)a
RNP-injected 20 hpi 6 5 (83.3) 5 5 (100)b
mRNA-injected 20 hpi 25 21 (84.0) 10 10 (100)b

Table 1.  Genome edition and mosaicism rates following the alternative protocols tested. Different superscript
letters indicate significant differences based on Chi-square test (p < 0.05).

The possible generation of more than two alleles per individual following CRISPR edition was initially
overlooked, as seminal work in mice did not report this phenomenon6, and because in this species breeding of
founders to obtain an heterozygous F1 generation is the routine protocol for experimental purposes. Under ideal
conditions, zygote genome edition should occur at the 2n2c stage, resulting in two indels (alleles). However,
DNA replication occurs soon after fertilization, before pronuclei fusion, transitioning to the 2n4c stage where
genome edition can result into more than two alleles, a phenomenon called genetic mosaicism. DNA edition may
also occur at later developmental stages (after cleavage) if both unedited alleles and active ribonucleoprotein are
present. Mosaic individuals are composed by more than one genetic cell type and have been consistently found
in most publications that have performed allele screening following CRISPR direct injection in zygotes of diverse
species such as mice7,8, pigs9–19, goats20, sheep21–23, cattle25 and rabbits26–34. In this article, we report strategies
to reduce genetic mosaicism following CRISPR edition of bovine embryos based on early delivery of CRISPR
components.

Results
Development of a shortened IVF protocol.  Conventional protocols of bovine in vitro fertilization (IVF)
entail the co-incubation of cumulus-oocyte complexes and spermatozoa for ~20 h35. Following co-incubation,
cumulus cells can be removed and then, the ooplasm of the presumptive zygotes can be clearly visualized and
microinjected. These IVF and micromanipulation conditions have been optimized to attain high developmental
rates, but the delivery of CRISPR components by microinjection at 20 h after the onset of IVF resulted in all edited
embryos being mosaic (Table 1). Aiming to explore the different possibilities of an earlier delivery of CRISPR
components, we first established the minimum gamete co-incubation time to achieve normal developmental rates
in a preliminary experiment. For this aim we reduced IVF time to 8, 9 or 10 h, observing that, in the case of the
semen used in these experiments, 10 h was the minimum gamete co-incubation time to achieve similar develop-
mental rates to the conventional 20 h IVF protocol (Fig. 1A,B and Table S1).

Characterization of S-phase in bovine zygotes.  Once the minimum co-incubation time was estab-
lished, we analysed the timing of DNA replication in bovine IVF derived zygotes from the minimum IVF time
(10 hours post-insemination –hpi-) to 20 hpi in 2 h intervals by 5-Ethynyl-2′-deoxyuridine (EdU) incorporation
(Fig. 1C). The data obtained show that DNA replication occurs short after fertilization, with ~40% of the zygotes
already replicating its DNA at 10 hpi (Fig. 1D). Four hours later (14 hpi) most zygotes were on S-phase and at the
time point when gamete co-incubation ceases in conventional IVF procotols (20 hpi), ~40% of the zygotes are on
S-phase. These kinetics suggest that S-phase spans for about 10 h and that by the end of the conventional gamete
co-incubation (20 hpi), most zygotes have replicated its DNA or are very close to end their S-phase. In this con-
text, an earlier CRISPR deliver is required to prevent the appearance of more than two alleles in a given embryo.

Developmental outcomes following different microinjection times.  Two early microinjection

strategies were tested: (1) microinjection right after a shortened IVF protocol (10 hpi) and (2) oocyte micro-
injection (0 hpi) followed by IVF of cumulus-free oocytes (Fig. 2A). Embryo developmental rates following
these alternative protocols were initially assessed without performing microinjection. As previously tested in
a smaller experiment (Fig. 1A,B), 10 hpi protocol yielded similar cleavage and blastocyst rate than the 20 hpi
control (Fig. 2B,C and Table S2). When oocytes were fertilized without cumulus cells (0 hpi group), cleavage rate
was reduced compared to both groups fertilized with cumulus cells (Fig. 2B, ANOVA p < 0.05), but no significant
differences were found on blastocyst yield between the three non-microinjected groups (Fig. 2C and Table S2). In
order to determine the developmental effects of the microinjection procedure and the possible toxicity of CRISPR
components without the interference of a possible detrimental effect of the genome edition per se, a genomic
target locus located in a non-coding region was chosen. The microinjection of CRISPR components (mRNA
and sgRNA at 0, 10 or 20 hpi and ribonucleoprotein at 0 hpi) in bovine oocytes or zygotes caused a statistically
significant reduction in cleavage and blastocyst rates in all four groups analysed compared to their correspond-
ing (time-matched) non-microinjected control (Fig. 2B,C, ANOVA p < 0.05). No significant differences were
observed in blastocyst rates between all microinjected groups (Fig. 2C and Table S2).

Genome edition and mosaicism rates following different microinjection times.  Genome edi-
tion rates were assessed in all four microinjected groups (3 microinjected with mRNA + sgRNA at 0, 10 and 20
hpi, and 1 microinjected with ribonucleoprotein at 0 hpi) and in 5 embryos obtained from a replicate microin-
jected with ribonucleoprotein at 20 hpi (Table 1). For this aim, a PCR product including CRISPR target site was

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Figure 1.  Developmental rates following shortened gamete co-incubation times and kinetics of S-phase in
bovine IVF zygotes. (A) Cleavage and (B) blastocyst rates obtained following gamete co-incubation for 8, 9, 10
or 20 h. (C) Representative pictures of S-phase analysis in bovine zygotes following EdU protocol; upper images
show a zygote that is not replicating its DNA (EdU negative), lower images depict a zygote on S-phase (EdU
positive). (D) Percentages of zygotes replicating its DNA from 10 to 20 hpi in 2 h intervals.

amplified and sequenced in at least 20 blastocysts per group. All groups displayed similar genome edition rates,
with more than 80% of the blastocyst produced from microinjected oocytes or zygotes being edited at the target
site. This sequencing strategy allowed to distinguish between edited and not-edited embryos, but does not pro-
vide the number of alleles harboured by a given embryo, as mixed sequencing peaks preclude allele identification
(Fig. 3). Allele identification was achieved by clonal sequencing, analysing 10 colonies per embryo in 20 embryos
on each of the three early microinjection groups (600 sequences) and 10 and 5 in 20 hpi group microinjected with
mRNA or ribonucleoprotein, respectively (150 sequences) (Fig. S1). Mosaicism rates were significantly reduced
in all early delivery groups compared to 20 hpi (70% reduction from 100% to ~30%, Table 1), but no differences
were noted between 0 or 10 hpi or between CRISPR injection formats. The number of embryos carrying WT (not
edited) alleles and carrying only alleles formed by indels not multiple of three (there would be KO embryos if the
target was allocated on a coding region) are shown in Table 2. As expected the percentage of embryos carrying
WT alleles was significantly higher in the group where all embryos were mosaic (20 hpi).

Genome edition and mosaicism rates targeting multiple coding loci.  To further test the most
user-friendly of the two alternative microinjection protocols developed (0 hpi), we analysed genome edition
and mosaicism rates in blastocysts derived from mRNA (n = 26) or ribonucleoprotein microinjections (n = 23)
targeting two coding loci simultaneously. These loci were localized at the beginning of the coding region of two
genes encoding for the proteins CSN2 and PAEP, both present in cow milk and responsible for allergic reactions
in humans. In this experiment genome edition rates were around or above 90%, and all embryos edited on one
locus were also edited in the other except for one in the RNP group (Table 3). Mosaicism rates were reduced to
12–24%, and again no statistically significant differences were found on edition, mosaicism or KO generation
rates between the groups injected with mRNA or ribonucleoprotein (Tables 3 and 4). Embryos were deemed as
KO for a gene when all alleles detected were formed by indels not multiple of three.

Discussion
Mosaicism constitutes a major problem for direct genome modification following the microinjection of CRISPR
microinjection in zygotes. The presence of more than two alleles in a given individual reduces greatly the chances
of KO generation in one step, without the need of subsequent breeding of founder individuals, as all alleles gener-
ated must disrupt the ORF of the target gene. The generation of a KO individual in a single pregnancy is especially
relevant in livestock species, where, in contrast to mice, generation intervals can be counted by years. In this per-
spective, the requirement of breeding mosaic founders for the generation of bi-allelic animals impede the culmi-
nation of genome modification projects within a reasonable time frame in these species. Besides, KO generation

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Figure 2.  Developmental rates following the alternative microinjection protocols tested. (A) Schematic
representation of the three alternative protocols tested and the conventional 20 hpi protocol. (B) Cleavage and
(C) blastocyst rates obtained following the alternative protocols tested with or without microinjection. Different
letters indicate significant differences based on ANOVA (p < 0.05).

in one step would allow to elucidate gene function during embryo development without the need of a colony of
heterozygous founders. A myriad of publications that have performed allele screening following CRISPR direct
injection in zygotes of diverse species such as mice7,8, pigs9–19, goats20, sheep21–23, cattle25 and rabbits26–34 have
encountered this problem and yet, probably due to the time and resource consuming allele screening, no study
has systemically analysed mosaicism rates following different protocols.
The analysis of S-phase on bovine zygotes show that DNA replicates short after fertilization, providing a phys-
iological explanation for the high mosaicism rates obtained following conventional microinjection times, as four
DNA copies of the target sequence will be present when genome edition occurs. The timing and duration of
S-phase in bovine in vitro produced zygotes overtly agrees with previous studies36,37, and although there are differ-
ences between bulls in both the onset of S-phase37 and cleavage division38, the protocols tested in this study could
be applied to the use of different sires with minor (10 hpi) or no (0 hpi) modifications. Likewise, early delivery
of CRISPR components is expected to reduce mosaicism rates in other species. In this context, earlier CRISPR
delivery has been observed to diminish mosaicism rates following CRISPR electroporation in mice39, and small
data sets from sheep24 and human40 studies also suggest a reduction.
The early microinjection protocols tested achieved a ~70–90% reduction in mosaicism rates. This reduction
constitutes a remarkable advance for direct KO generation, but mosaicism was not completely abolished. The
use of ribonucleoprotein instead of RNAs did not reduced further mosaicism rates, suggesting that the time
required for RNA translation and ribonucleoprotein assembly was not a crucial factor for mosaicism appearance.

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Figure 3.  PCR and clonal sequencing of an edited non-mosaic embryo. Upper image show the sequencing
reaction of a PCR product, mixed peaks indicates edition but do not allow allele discrimination. Lower images
show the sequencing reaction of individual alleles following clonal sequencing.

No. of edited No. of edited non- No. of embryos containing No. of embryos containing only
Group embryos mosaic embryos (%) an unedited WT allele (%) indels non-multiple of 3 (%)
RNP-injected 0 hpi 20 14 (70.0)a 8 (40.0)a,b 3 (15.0)
mRNA-injected 0 hpi 20 14 (70.0)a 6 (30.0)a 6 (30.0)
mRNA-injected 10 hpi 20 13 (65.0)a 6 (30)a 6 (30.0)
RNP-injected 20 hpi 5 0 (0)n.d. 4 (80.0)n.d. 0 (0)
mRNA-injected 20 hpi 10 0 (0)b 8 (80.0)b 1 (10.0)

Table 2.  Percentage of embryos containing unedited WT alleles or only indels non-multiple of 3. Different
superscript letters indicate significant differences based on Chi-square test (p < 0.05) between the four
microinjection groups.

No. of embryos genotyped by No. of embryos No. of mosaic embryos No. of embryos genotyped by No. of embryos No. of mosaic embryos
Group PCR sequencing for PAEP edited for PAEP (%) for PAEP (%) PCR sequencing for CSN2 edited for CSN2 (%) for CSN2 (%)
RNP-injected
23 21 (91.3) 3 (14.3) 23 20 (87.0) 3 (15.0)
0 hpi
mRNA-injected
26 25 (96.2) 3 (12.0) 26 25 (96.2) 6 (24.0)
0 hpi

Table 3.  Genome edition and mosaicism rates following dual targeting (PAEP and CSN2) by mRNA or RNP
microinjections at 0 hpi. No significant differences were observed based on Chi-square test (p > 0.05).

Somehow surprisingly, CRISPR delivery to bovine oocytes (i.e., before fertilization) did not decrease mosaicism
rates further compared to the delivery to presumptive zygotes right after a reduced IVF protocol (10 hpi), sug-
gesting that CRISPR activity is very low before pronuclei formation. A possible explanation for this result is that
CRISPR may not be able to recognize its target locus before some degree of chromatin decondensation has been
achieved in the pronuclei, as chromatin accessibility exerts a great impact on Cas9 binding in vivo41,42. In this
sense, the high degree of DNA condensation in matured oocytes and especially on spermatozoa43 may impede an
earlier CRISPR-mediated genome edition.
In conclusion, early delivery of CRISPR components to bovine oocytes prior to IVF or zygotes following a
shortened IVF reduces mosaicism rates from 100% to ~10–30% while achieving similar genome edition and
developmental rates. Oocyte microinjection is more convenient schedule-wise than microinjection following a 10
h IVF protocol and both RNA or ribonucleoprotein delivery formats can be used, as both achieved similar results.

Methods
Bovine in vitro production.  Ovaries were collected at local slaughterhouse and transported to the lab-
oratory within 2 h. Cumulus-oocyte complexes (COCs) were collected from 2 to 8 mm diameter follicles and
selected based on conventional morphological criteria44. In vitro maturation (IVM) was performed in TCM-199
supplemented with 10% (v/v) fetal calf serum (FCS) and 10 ng/ml epidermal growth factor at 39 °C under an
atmosphere of 5% CO2 in air with maximum humidity for 24 h. IVF was performed with frozen-thawed sperma-
®
tozoa from a single sire selected by Bovipure (Nidacon). Spermatozoa were diluted to a final concentration of
106 spermatozoa/ml and were co-incubated with mature oocytes in TALP medium at 39 °C under an atmosphere

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No. of embryos No. of KO embryos No. of embryos No. of KO embryos No. of embryos edited No. of KO embryos for
Group edited for PAEP for PAEP (%) edited for CSN2 for CSN2 (%) for both PAEP and CSN2 both PAEP and CSN2 (%)
RNP-injected
21 11 (52.3) 20 8 (40.0) 20 5 (25.0)
0 hpi
mRNA-injected 0 hpi 25 8 (32.0) 25 14 (56.0) 25 5 (20.0)

Table 4.  Percentage of KO embryos (i.e., harbouring only frame-disrupting alleles) following dual targeting
(PAEP and CSN2) by mRNA or RNP microinjections at 0 hpi.

of 5% CO2 in air with maximum humidity. Different co-incubation times were initially tested (8, 9, 10 and 20 h)
to determine the minimum co-incubation time (>80 embryos/group in two independent replicates). For micro-
injection experiments, gametes were co-incubated for 10 (10 hpi group) or 20 h (0 and 20 hpi groups) (Fig. 2A).
Microinjected and non-microinjected oocytes of 0 hpi groups were denuded by vortexing in PBS supplemented
with 300 µg/ml hyaluronidase for 3 min prior to fertilization, in order to visualize the ooplasm membrane for
microinjection. In 10 and 20 hpi groups intact COCs were used for IVF and cumulus cells were removed at the
end of the co-incubation time by vortexing in PBS for 3 min. Cumulus-free presumptive zygotes were cultured in
vitro in SOF media supplemented with 5% FCS under an atmosphere of 5% CO2 and 5% O2 in air with maximum
humidity. Cleavage rates were assessed at 48 hpi and blastocyst yield was recorded 9 days post-insemination (dpi)
in 3–4 independent replicates per group. Statistical differences in developmental rates were assessed by One Way
ANOVA using SigmaStat software.

Assessment of DNA replication in bovine zygotes.  DNA synthesis was detected by the incorporation
of 5-Ethynyl-2′-deoxyuridine (EdU) (Click-iT EdU HCS assay kit, Invitrogen). Briefly, following 10, 12, 14, 18
and 20 hpi, 243 presumptive zygotes from 3 independent replicates were vortexed 3 min to remove cumulus cells
and incubated in 50 µM EdU for 30 min45. EdU labelling was detected following manufacturer instructions and
pronuclei were counterstained with 10 µg/ml Hoechst. Zygotes were observed on an epifluorescence inverted
microscope (Nikon Eclipse TE300) and considered to be on S-phase stage when they displayed at least one pro-
nucleus labelled with EdU.

CRISPR/Cas9 microinjection.  Details for sgRNAs are provided on Table S3. sgRNAs were synthesized and
®
purified using Guide-it sgRNA In Vitro Transcription Kit (Takara). Capped polyadenylated Cas9 mRNA was
®
produced by in vitro transcription (mMESSAGE mMACHINE T7 ULTRA kit , Life Technologies) using as tem-
plate the plasmid pMJ920 (Addgene 42234) linearized with BbsI and treated with Antarctic phosphatase (NEB).
mRNA was purified using MEGAClear kit (Life Technologies). A solution of 300 ng/µl of mRNA and 100 ng/µl
of each sgRNA was used for RNA microinjection. For ribonucleoprotein injection, Guide-it Recombinant Cas9
(Takara) and sgRNA/s were mixed to a final concentration of 300 ng/µl and 60 ng/µl, respectively, and incubated
at 37 °C for 5 min to achieve ribonucleoprotein assembly prior to microinjection. Previous experiments were con-
ducted to determine that the concentrations used did not reduce developmental rates compared to sham (buffer)
injections. Microinjection was performed under a Nikon Diaphot TMD inverted microscope delivering 3–5 pl
into the ooplasm using a filament needle.

Embryo genotyping.  CRISPR-injected embryos were kept in culture until day 9 after insemination. Zona
pellucida was removed from unhatched blastocysts by incubation in acid PBS (pH 2) to avoid any residual sper-
matozoa and to facilitate subsequent enzymatic digestion. Zona-free blastocysts were individually placed at the
bottom of a 0.2 ml PCR tube and stored at −80 °C until analysis. Each blastocyst was digested in 8 µl of Picopure ®
(ThermoFisher Scientific) for 1 h at 65 °C followed by inactivation at 95 °C for 10 min.
Genotyping of the intronic region was performed by clonal sequencing. PCR was performed on a 50 µl
reaction containing 4 µl of the inactivated digestion product under the following conditions: 95 °C for 2 min;
39 × (95 °C for 20 s, 60 °C for 30 s, 72 °C for 40 s); 72 °C for 5 min; hold at 8 °C. PCR was performed using primers
spanning the target sequence (Table S4). The PCR product from each blastocyst was purified using FavorPrep ™
PCR Purification Kit (Favorgen). The purified product was Sanger sequenced and analyzed for the presence of
indels. Consecutively, the purified PCR products from edited embryos, identified by a mixed sequence reaction
around the target site (Fig. 3), were analyzed by clonal sequencing. For that aim, the purified PCR product was
ligated into pMD20 T-vector (Takara) by Blunt/TA Ligase (NEB) and transformed into Escherichia coli DH5-α
competent cells. For each embryo analysed, ten plasmids containing the insert were Sanger sequenced.
Genotyping of PAEP and CSN2 target sequences was performed by deep sequencing. A first 30 cycle amplicon
PCR was performed on 25 µl reaction containing 2 µl of the inactivated digestion product using the conditions
detailed above and primers including Illumina overhangs (marked in italics in Table S4). Amplicons were purified
by AMPPure XP beads (Beckman Coulter) and libraries were prepared by an index PCR which added Illumina
adaptors and indexes identifying each embryo using Nextera XT (Illumina). Libraries were purified, pooled to
2 nM and sequenced on Illumina miSeq platform providing 250 bp paired-end sequencing reads. Individual
alleles were identified following QC filtering, mapping to reference and variant calling.
Embryos showing more than two alleles by clonal or deep sequencing were considered mosaic. Statistical
differences in genome edition and mosaicism rates were assessed by Chi-square test using SigmaStat software.

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Acknowledgements
We thank the slaughterhouse “Transformación Ganadera de Leganés SA” for gently providing bovine ovaries to
conduct the experiments. This work has been funded by the projects RYC-2012-10193, AGL2014-58739-R and
AGL2017-84908-R from the Spanish Ministry of Economy and Competitiveness (MINECO) and the project
StG-757886-ELONGAN from the European Research Council. I.L.T. is funded by a FPI fellowship by MINECO.

Author Contributions
I.L.T., B.G.C., F.C.A., J.C.F., O.O. and P.B.A. participated on in vitro embryo production and embryo genotyping.
I.L.T. performed oocyte/zygote microinjections. P.B.A. and I.L.T. performed S-phase analysis, statistical analysis
and wrote the manuscript.

Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-51366-8.
Competing Interests: The authors declare no competing interests.
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