Maintenance of mouse lines
All mouse lines (Mus musculus) were maintained under UK Home Office Regulations, UK Animals (Scientific Procedures) Act 1986, and according to ethical guidelines at the Francis Crick Institute. Permission for animal experiments was granted by The Crick Biological Research Facility Strategic Oversight Committee (BRF-SOC) incorporating Animal Welfare and Ethical Review Body (AWERB) (Project Licence P8ECF28D9). All mice were kept in individually ventilated cages (IVC), with constant access to food, automatic watering systems, and air management systems which control air flow, temperature (20–22 °C) and humidity (49–62 %). The mice were kept under a 12 h light and 12 h dark cycle. The mouse lines were checked daily and maintained in specific pathogen-free (SPF) conditions. Sufficient nesting material and environmental enrichment was provided. XTop1Y mice were generated on a 129P2/OlaHsd background, in order to take advantage of the GenOway Quick Knock-in approach. After generating a stable line, the XTop1Y mice were maintained on C57BL/6J background in order to more closely recapitulate the genetic background of the other transgenic lines. The H11Top1 mouse line was generated on a C57BL/6J background and maintained on a C57BL/6J background. XCas9Y and XYCas9 mice were generated and maintained on a C57BL/6 N background. Wild-type control mice were C57BL/6J. 6–10-week-old female and 8–14-week-old male mice were used for matings.
Embryonic stem cell derivation and maintenance
Embryos were collected at E3.5 by flushing the uterus with Follicle Holding Medium (FHM) from timed mating 6–8-week-old females, and placed in individual wells of a 24-well plate with 500 μl of 2i/LIF. Outgrowths were dissociated and mESCs seeded into a 4-well plate in 2i/LIF. mESCs were passaged by removing 2i/LIF, washing with PBS, followed by dissociation with TrypLE (Gibco), quenching with 2i/LIF and pipetting into a single cell suspension. Following centrifugation at 200 g for 3 min, mESCs were resuspended and seeded in new plates52. mESC lines were maintained in 2i/LIF conditions on laminin-coated tissue culture grade plasticware53.
Primer design
All primer pairs used in this study were designed using the publicly available tool Primer3 (http://bioinfo.ut.ee/primer3/). All PCR amplification was carried out using Q5 High-Fidelity DNA polymerase (NEB) at recommended Q5 thermocycling conditions. To amplify Top1 and Atm target exons for MiSeq analysis, primers were designed using Primer3 and extended to contain MiSeq adaptor sequences (see relevant section). All primer sequences are listed in Table S1.
sgRNA design
All sgRNAs were designed using publicly available in silico tools54. Single sgRNAs with a predicted high on-target activity and low off-target activity were selected. Oligonucleotides with BbsI overhangs were annealed and ligated into the relevant vector, according to a published protocol55. For transient electroporation of sgRNAs, the sgRNA oligonucleotide was bought from Integrated DNA Technologies. All sgRNA sequences are contained in table S1.
Fluorescence-activated cell sorting (FACS)
Transfected mESCs were dissociated using TrypLE into a single cell suspension, centrifuged at 200 × g for 3 min, and resuspended in sorting media (2% FBS in 2i/LIF). mESCS were filtered (40 μM) and sorted using the Aria Fusion Flow Cytometer with a 100 μM nozzle. mESCS were firstly gated on forward and side scatters properties, followed by gating on either eGFP-mCherry double-positive expression or eGFP only. The eGFP-only population acted as the CRISPR-Cas9 negative control.
Embryo dissociation and flow cytometry
E9.5–E12.5 embryos were dissociated and prepared for flow cytometry according to a previously published protocol56. Dissociated cells were filtered (40 μM) and kept on ice in sterile PBS with 2% FBS prior to flow cytometry and analysis on the MACSQuant VYB. Single cells were analysed on forward and side scatter properties, followed by gating on eGFP or mCherry expression (Table S4.) using FlowJo v10.7 software. Gating strategies for all the embryo samples for either eGFP expression or mCherry expression can be seen in Supplementary Fig. 8 and Supplementary Fig. 9, respectively.
Generating the XTop1 mouse line
The Top1 sgRNA targeting vector was generated using plasmid pX333 as a backbone (Addgene #64073)57. The Cas9 cassette was replaced with an mCherry reporter. Top1 sgRNA2 were inserted using BbsI55. The resulting hU6-sgRNA2-pCbh-mCherry construct was used to generate the XTop1 transgene by GenOway (Lyon, France) using their Quick Knock-in approach in E14Tg2a mESCs derived from 129P2/OlaHsd. Human Hprt exons 1 and 2 were inserted into the sgRNA2-mCherry targeting vector. The construct was targeted to the Hprt locus in mESCs that lacked exons 1 and 2. Upon recombination of the targeting vector into Hprt, gene function was restored. Correctly targeted clones were selected with hypoxanthine-aminopterin-thymidine (HAT) medium. Eight HAT-resistant clones were selected and amplified for confirmation of on-target integration. X- integration clones were confirmed using PCR and Sanger sequencing for validation.
Generating the H11
Top1 mouse line
To generate the H11Top1 targeting vector we repurposed the same hU6-sgRNA2-pCbh-mCherry plasmid used to generate XTop1. The human U6 promoter-sgRNA cassette and pCbh-mCherry reporter sequence was inserted into the TARGATT MCS plasmid vector #358,59 (Applied Stem Cell). The TARGATT plasmid containing hU6-sgRNA2-pCbh-mCherry was microinjected into attPx3 embryo pronuclei with ϕC31 integrase (sourced from Applied Stem Cell protocols via InsightBio). The plasmid DNA was diluted to 6 ng/μl in 5 mM Tris/0.1 mM EDTA. 10 μl plasmid was mixed with 10 μl TARGATT integrase solution and filtered through a 0.2 μM syringe filter immediately prior to zygotic injection. Embryos were surgically transferred into pseudopregnant females. Founders were screened by in vivo fluorescence imaging at 3-4 days post birth using the IVIS Lumina XR (Caliper LifeSciences) with Living Image 4.4 software, excitation filter at 535 nm and emission filter dsRed. One founder (n = 1/25) was mCherry positive and germline transmitted.
Generating the XCas9 mouse line
The Cas9-eGFP targeting vector was generated using the pX330 (Addgene #42230)60 plasmid backbone, containing a pCAG driven 3X FLAG-NLS-Cas9-T2A-eGFP construct. X chromosome homology arms, amplified from C57BL/6J DNA, and a LoxP-flanked pPGK-Neomycin cassette were inserted using directional cloning or Gibson Assembly (NEBuilder HiFi DNA Assembly Cloning Kit). C57BL/6N mESCs were maintained in serum/LIF conditions and transfected with the Cas9-eGFP targeting vector plasmid and an sgRNA targeting Hprt exon 2 using Lipofectamine 2000, according to manufacturer’s instructions. Targeted mESC clones were selected by G418 (270 mg/ml) for 8–10 days. Forty-eight surviving clones were picked into a 96-well plate and expanded. PCR genotyping was performed on extracted DNA in a total volume of 25 μl (12.5 μl NEB Q5 High-Fidelity Master Mix, 10 mM each primer), using primer forward and reverse pairs aligning to the endogenous Hprt locus and to the transgene construct. Resultant PCR amplicons were analysed by gel electrophoresis for corresponding to the expected amplicon size, and by Sanger sequencing. Of 48 clones, 9 were found to be successfully targeted (19%). Targeted mESC clones were injected into albino C57BL/6J-Tyr<c-Brd> blastocysts and surgically transferred into pseudopregnant females. XCas9Y mESC contribution to founders was assessed by coat colour. High contribution transgenic males were bred with C57BL/6J-Tyr<c-Brd> albino females, and offspring with black coat colour were genotyped for the transgene to confirm germline transmission.
Generating the YCas9 mouse line
In order to generate XYCas9, we repurposed the Cas9-eGFP plasmid vector used to generate the XCas9Y mouse model. The Cas9-eGFP plasmid contained a pCAG driven 3X FLAG-NLS-Cas9-T2A-eGFP construct. The Cas9-eGFP cassette was targeted to the Y-linked Uty locus, which is ubiquitously expressed. In order to maintain Uty expression, the Cas9-eGFP plasmid was targeted to Uty exon 17, which is retained in all Uty isoforms. A plasmid vector containing exons 19-30 as cDNA (exon 18 is excised during normal Uty transcription) was inserted into the plasmid vector upstream of an IRES cassette, Following the IRES was Cas9-eGFP. The plasmid was electroporated into C57BL/6N mESCs following a proprietary protocol carried out by GenOway (Lyon, France). Clones underwent positive neomycin selection and negative DTA selection. 24 positive clones were selected by a pre-screen and five were confirmed positive Y-integration clones by PCR and Sanger sequencing. Following this result, GenOway also performed sequencing analysis spanning the Y-integration region, and concluded that four positive clones were correctly targeted.
Genotyping
Pups were genotyped using assays for the Cas9-eGFP transgene, the mCherry transgene, and sexed by the presence of Y-linked gene Sry using Transnetyx assays. XCas9 hemizygous versus homozygous females were distinguished by genotyping for the Hprt exon 2 deletion (primer sequences available in Table S1.). Thermocycling conditions as follows: 95 °C (3 min), 30 X cycles of 95 °C (20 s), 60 °C (15 s), 72 °C (20 s) followed by 72 °C (3 min) and 4 °C (hold). The XO female generated from breeding XCas9Y males with H11Top1 homozygous females, was characterised by DNA extraction from ear biopsy tissue (see relevant methods); X-chromosome and transgene copy number analyses using digital droplet PCR (see relevant methods), and low-pass whole genome Oxford Nanopore Technologies sequencing (see relevant section). H11Top1 hemizygous versus homozygous mice were distinguished using mCherry copy number analysis using Transnetyx raw data.
MiSeq high throughput sequencing and indel analysis
Lysis was performed using lysis buffer (10X KT buffer, 10% NP40) with proteinase K (1 mg/ml) digestion. Target Top1 exons were PCR-amplified using MiSeq PCR primers (Table S1.) in a total volume of 25 μl (12.5 μl NEB Q5 High-Fidelity Master Mix, 5 mM each primer) Correct PCR amplification was confirmed by gel electrophoresis. Resultant PCR amplicons were purified using solid-phase reversible immobilisation (SPRI) beads (according to manufacturer’s protocol)61 and resuspended in 15 μl nuclease-free water. To prepare libraries, 1 μl DNA was PCR amplified with NXT Primer Mix (IDT-8nt) using Q5 High-Fidelity Master Mix (NEB). Thermocycling conditions as follows: 95 °C (3 min), 10 X cycles of 95 °C (30 s), 55 °C (15 s), 72 °C (30 s) followed by 72 °C (5 min) and 12 °C (hold). PCR products were purified using SPRI bead clean. Libraries were quality controlled using Glomax (Promega, manufacturer’s procedure) and pooled. Pooled libraries were quantified using Qubit Lifetech and HSD1000 Tapestation Agilent. MiSeq libraries were sequenced on Illumina MiSeq-Nano 250PE to generate paired-end (2 × 250 bp) sequencing reads. Resultant reads were demultiplexed and fastq files were collapsed using FastX Toolkit (v0.0.13) [https://github.com/agordon/fastx_toolkit]. To assess the rate of indel-production by CRISPR-Cas9, the reads were aligned to the mouse reference genome mm10 with the Burrows-Wheeler Alignment tool (BWA, v0.7.170)62 using the mem algorithm with default settings and then analysed using the R package CrispRVariants (v1.14.0)63. Scripts are deposited on github [https://github.com/jzohren/crispr-miseq]. Raw MiSeq read counts are available in the associated Source Data files. In each experiment, SNV; single nucleotide variant, I; insertion, D; deletion. The plus/minus number refers to the position that the mutation has occurred, relative to three nucleotides upstream of the PAM. The second number refers to the number of nucleotides that have been inserted or deleted. Data will be made available upon reasonable request.
Low-pass whole genome Oxford Nanopore Technologies sequencing
DNA was extracted using the phenol-chloroform method as described previously64. DNA libraries were prepared in accordance with the Oxford Nanopore Technologies (ONT) SQK-LSK109 Ligation Sequencing protocol, with multiplexing using the EXP-PBC096 kit. Libraries were sequenced on a FLO-MIN106D R9.4.1. flow cell on the MinION MIN-106B. Basecalling was performed using ONT-Guppy v3.2, and data was mapped using minimap2 v2.1765 and SAMtools v1.966, and analysed using base R v4.0.2 and Excel. Raw chromosome read counts are available as Supplementary Table 5 and also available as Source Data. Data will be made available upon reasonable request.
Quantitative PCR analysis
RNA was extracted using TRI Reagent (Sigma-Aldrich), according to the manufacturer’s protocol. cDNA was synthesised using the Thermo Scientific First Strand cDNA Synthesis Kit, according to the manufacturer’s protocol. Samples were analysed in triplicate, in 10 μl total volume (5 μl TaqMan 2X Universal PCR Master Mix, 0.5 μl TaqMan probe, 2.5 μl nuclease-free water, 2μl cDNA). Thermocycling conditions as follows: 95 °C (10 min), 40 X cycles of 95 °C (15 s), 60 °C (1 min), followed 4 °C (hold). Resulting ddCt values were calculated by normalising to Gapdh expression from C57BL/6 samples unless otherwise described. TaqMan probes used are available in Table S2.
Digital droplet qPCR
DNA was extracted by phenol-chloroform precipitation. Digital droplet qPCR (ddPCR) reactions were performed in 20 μl total volume with 20 ng DNA, according to manufacturer’s instructions (Bio Rad ddPCR Supermix for Probes). The ddPCR was performed on a Bio-Rad PCR machine and analysed using QuantaSoft v1.7.4.0917. ddPCR was performed using Taqman copy number probes and are available in table S2. Thermocycling conditions as follows: 95 °C (10 min), 39 X cycles of 94 °C (30 s), 60 °C (1 min), followed by 98 °C (10 min) and 4 °C (hold).
Protein extraction and western blot
Protein was extracted from samples using 1X RIPA buffer with additional phosphatase and protease inhibitors, and PMSF. Upon adding protein extraction buffer to samples, samples were kept on ice for 30 m, the following centrifugation at 5900 × g at 4 °C for 10 m. The supernatant was collected and protein quantified using a bicinchoninic acid (BCA) assay and analysed using Kaleido 2.0. Proteins were separated using PAGE and transferred to 0.45 μm pore Nitrocellulose membrane (Amersham Protran). Membranes were blocked with 5% skimmed milk/TBST for 1 h at room temperature and incubated with primary antibodies overnight at 4 oC. CAS9 (Novus Bio) and TOP1 (Abcam) antibodies were used at 1:500, α-TUBULIN (Sigma) at 1:2000, GAPDH (Santa Cruz Biotechnology) at 1:3000 dilutions. Secondary antibodies conjugated to HRP were used (anti-mouse IgG-HRP (Santa Cruz, 1:4000 dilution), or anti-rabbit IgG-HRP (Cell Signalling 1:4000 dilution)) and signals were detected using Clarity Western ECL Substrate (Bio-Rad). Antibodies used are available in table S3. Raw blots are available as Source Data.
Southern blot
DNA was extracted by phenol-chloroform precipitation, digested using appropriate restriction enzymes, and phenol-chloroform precipitation repeated. DNA was loaded onto a 1% agarose gel and gel electrophoresis run overnight at 29 V, followed by the addition of bromophenol blue, and further running at 50 V for 2–3 h. Following gel electrophoresis, the agarose gel was treated by washing in depurination (0.25 M HCl), denaturation (1.5 M NaCl, 0.5 M NaOH) and neutralisation (1.5 M NaCl, 0.5 M Tris pH 7.5) buffers and overnight blotting onto a positively charged nylon membrane. After blotting, the DNA was fixed by UV crosslinking (1200U joules, 2 m) and drying. The membrane then underwent hybridisation to the Neomycin probe (Fig. S7, Table S1), produced according to manufacturer’s instructions (Roche DIG probe synthesis kit) and incubation overnight in a hybridisation oven at the optimal temperature (48 °C for Neomycin). Post-hybridisation, the membrane was washed (2X SSC, 0.1% SDS) at room temperature, and at 65 °C (0.1X SSC, 0.1% SDS). Following this, the membrane was blocked with blocking buffer and incubated for 30 m at room temperature with anti-DIG antibody (1:40,000; Roche DIG Luminescent Detection Kit; Table S3.), washed (maleic acid, 0.3% tween-20), and exposed to CSPD in detection buffer under darkness before film development. Raw blots are available as Source Data.
Embryo electroporations and post-natal Atm mutation analysis
Zygote stage embryos were generated by in vitro fertilisation with either XCas9Y or XYCas9 males and wild-type females. Zygotes were washed through droplets of opti-MEM prior to electroporation with Nepagene 21 (5 mm). sgRNAs were prepared to 12 μM final concentration and brought to room temperature for 10 mins immediately prior to addition to electroporation mix. Fifty microliters of electroporation/RNA mix was added into the electroporation chamber and impedance measured to be at 0.48–0.52 kOhm. Embryos were transferred into pseudo-pregnant methods and left to litter down. At 2 weeks, pups were ear biopsied, DNA extracted, and MiSeq PCRs performed (see relevant methods sections). Daughters from XCas9Y fathers showed mutation efficiencies of 99.7, 99.4, 98.4, 98.8, 99.3, 99.7, and 99.8%. Non-mutant brothers showed a mean mutation efficiency of 0.18% ± 0.19 s.d (n = 24 males). Sons from XYCas9 fathers showed mutation efficiencies of: 99.8, 99.6, 78.3 and 51.9% (electroporation round 1), and 99.9, 99.9, 99.9, 99.9, 99.9, 85.3, 99.9, 99.9, 92.1, 73.7, 99.9, 99.9, 99.9, 99.9, 100, 99.9, 99.9 and 99.9% (electroporation round 2). Non-mutant sisters showed a mean mutation efficiency of 0.04% ± 0.03 s.d (electroporation round 1, n = 5 females) and 0.03% ± 0.02 s.d (electroporation round 2, n = 20 females).
Ovary section and immunofluorescence
Ovaries were collected at post-natal week 8 for wild-type and Atm wild-type females and fixed in 4% paraformaldehyde (PFA) overnight at room temperature. Fixed ovaries were washed with phosphate-buffered saline (PBS), embedded in paraffin and sectioned at 6 μM. For immunofluorescence, slides were deparaffinised and rehydrated by a series of xylene and ethanol, prior to antigen retrieval (0.1 M sodium citrate, 30 min). Sections were blocked for 15 min (5% bovine serum albumin, BSA, in PBS/Tween 20), and incubated with a germ cell marker primary antibody at room temperature for 1 h (rabbit anti-MVH; 1:100; antibodies listed in Table S3), followed by incubation with a secondary antibody (Alexa Fluor® 594, dilution 1:200) at room temperature for 1 h. Slides were mounted with Vectashield plus DAPI (Thermo-Fisher). Images were obtained using Olympus upright BX63 microscope with associated robotic slide loader with an Excite Exacte Measured Metal halide source and built-in Koehler illumination for transmitted light. Captured images were analysed using QuPath v0.2.3 (Open Source).
Follicle quantification
Per mouse, one ovary and every sixth section per ovary was counted for the presence of primordial, primary, secondary and antral follicles (considered as total in Fig. 4).
Immunofluorescence of testis nuclear spreads
Testis were collected at post-natal week 8 for wild-type and Atm mutant males. Glass slides (ThermoFisher AA00008032E00MNT10) were boiled in water for 10 min and dried completely. Testes were dissociated in Roswell Park Memorial Institute (RPMI) medium. 100 μl of cell suspension was transferred to each slide, with the addition of 50 μl 0.05% TritonX-100 (in water) and kept at room temperature for 10 min. Cells were then fixed in 2% PFA/0.02% sodium dodecyl sulfate (SDS) (in PBS) at room temperature for 1 h, washed with water, and dried. Slides were blocked (0.15% BSA/0.1% Tween20 in PBS) at room temperature for 1 h. Slides were incubated with primary antibodies (SYCP3 1:100 and γH2AX 1:250; antibodies listed in Table S3) in a humidified chamber at 37 oC overnight. Secondary antibodies (Alexa Fluor® 488 and Alexa Fluor 568) were applied in blocking buffer at 37 oC for 1 h. Samples were then washed in PBS at room temperature and mounted in Vectashield with DAPI. Images were taken using Deltavision Microscopy System (100x/1.35NA Olympus UPlanApo objective; GE Healthcare).
Testis histology
Testis were collected at post-natal week 8 for wild-type and Atm mutant males and fixed overnight in Bouin’s solution. Fixed samples were washed in 70% ethanol, embedded in paraffin, sectioned, and stained with Periodic Acid-Schiff staining. Images were taken using Olympus BH2 microscope with 40x/0.70NA Olympus SPlan objectives.
Litter size quantification
Every experimental and control mating female pregnancy was recorded and females were checked for pups that had been born. Pups that survived to weaning stage were ear biopsied for genotyping at ~14 days post birth and counted for litter size measurements. Pups that died prior to weaning were genotyped if tissue was available. Pregnancies that produced pups that died shortly after birth but tissue could not be recovered (e.g., cannibalisation) were counted but not included in total litter size: XTop1Y x R26-Cas9 = 0, XTop1Y x Wildtype = 1, H11Top1 x R26-Cas9 = 0, H11Top1 x Wildtype = 0, XCas9Y x H11Top1 = 3, XCas9Y x Wildtype = 2, XYCas9 x H11Top1 = 0, XYCas9 x Wildtype = 0. Total litter losses (no pups found after a successful pregnancy) were counted: XTop1Y x R26-Cas9 = 2, XTop1Y x Wildtype = 2, H11Top1 x R26-Cas9 = 0, H11Top1 x Wildtype = 1, XCas9Y x H11Top1 = 1, XCas9Y x Wildtype = 3, XYCas9 x H11Top1 = 3, XYCas9 x Wildtype = 1.
Statistical analysis
To determine if the number of male or female pups born from experimental matings deviated from an expected 50:50 Mendelian frequency a Chi-squared test was performed. Expected was considered an equal 50:50 male:female ratio, and Observed was the quantified male and female number. Sample size was considered to be sufficient number of pups to accurately make the assumption that single-sex litters could not be down to random chance. To determine if mutation efficiency in mCherry-eGFP samples deviated from negative control eGFP only samples, a Mann–Whitney test for non-parametric data was performed. To determine the difference in average litter size between experimental and control samples, a Mann–Whitney test for non-parametric data was performed.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
