Technical Report | Published:

CRISPR–Cas9-mediated base-editing screening in mice identifies DND1 amino acids that are critical for primordial germ cell development

Abstract

CRISPR-mediated base editing can introduce single-nucleotide changes in the DNA of living cells. One intriguing application of base editing is to screen pivotal amino acids for protein function in vivo; however, it has not been achieved. Here, we report an enhanced third-generation base-editing system with extra nuclear localization sequences that can efficiently introduce a homozygous base mutation in embryonic stem cells. Meanwhile, we establish a strategy to generate base-mutant mice by injection of haploid embryonic stem cells carrying a constitutively expressed enhanced third-generation base-editing system (4B2N1) and single guide RNA into oocytes. Moreover, transfection of 4B2N1 cells with a single guide RNA library targeting the Dnd1 gene allows one-step generation of mutant mice with a base mutation. This enables the identification of four missense mutations that completely deplete primordial germ cells through disruption of DND1 protein stability and protein–protein interaction. Thus, our strategy provides an effective tool for in vivo screening of amino acids that are crucial for protein function.

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Data availability

The deep-sequencing and whole-exome sequencing data from this study have been deposited in the Gene Expression Omnibus (GEO) under the accession codes GSE115015 and GSE115017. Images corresponding to the three independent replicates for Fig. 2a have been deposited in Figshare (https://doi.org/10.6084/m9.figshare.7387607.v1). The source data for Figs. 1c, 2b and 5e and Supplementary Fig. 6d have been provided as Supplementary Table 13. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Competing interests

The authors declare no competing interests.

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Change history

  • 07 December 2018

    In the version of this Article originally published, Supplementary Figure 2 was incorrectly linked to Supplementary Figure 3, Supplementary Figure 3 was incorrectly linked to Supplementary Figure 4, Supplementary Figure 4 was incorrectly linked to Supplementary Figure 5, Supplementary Figure 5 was incorrectly linked to Supplementary Figure 6, Supplementary Figure 6 was incorrectly linked to the first page of Supplementary Figure 7, and Supplementary Figure 7 was incorrectly linked to only the second page of Supplementary Figure 7. The files have now been replaced to rectify this.

  • 04 February 2019

    In Fig. 2a of this Technical Report originally published, the authors inadvertently used the same set of images for the 4B2N1 and 4B2N3 cells when preparing the figure. The three images (bright field, Oct4-EGFP and pCAG-mRFP) of 4B2N3 cells have now been replaced with the correct versions. The source data for the four cell lines in Fig. 2a, captured in the three independent experiments, have been deposited to Figshare (https://doi.org/10.6084/m9.figshare.7387607.v1), and the figure legends and Methods section have been amended to reflect this. Additionally, the unprocessed blots in Supplementary Fig. 7 corresponding to the top right ‘WCL IB: Flag’ panel of Fig. 7e were mistakenly duplicates of the unprocessed blots for the bottom left ‘IP Flag IB: HA’ panel of Fig. 7e, and all unprocessed blots for Supplementary Fig. 6 were mislabelled as blots corresponding to Supplementary Fig. 7. Supplementary Fig. 7 has now been updated to show the correct unprocessed blots for the bottom left ‘IP Flag IB: HA’ panel of Fig. 7e and to correct the labelling of the unprocessed blots corresponding to Supplementary Fig. 6.

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Acknowledgements

This study was supported by the Genome Tagging Project and grants from the Chinese Academy of Sciences (XDB19010204, OYZDJ-SSW-SMC023 and Facility-based Open Research Program), the National Natural Science Foundation of China (31530048, 81672117 and 31730062) and Shanghai Municipal Commission for Science and Technology (16JC1420500, 17JC1400900, 17JC1420102 and 17411954900).

Author information

Q.L. developed the enhanced BE3 system and designed the sgRNA library. Y.L. and X.L. purified the proteins and analysed protein stability and protein–protein interaction. Q.L., S.Y. and Q.Y. performed mouse embryo microinjection and embryo transplantation. Q.L., S.H. and L.L. derived cell lines and characterized cells. Q.L. and S.H. performed genotyping and DNA sequencing analyses. Q.L. and Y.D. dissected E8.5, E9.5 and E12.5 embryos and analysed PGCs in gonads. W.T. helped with the mouse experiments. H.S. helped with the PGC experiments. M.Y. and Z.S. analysed the whole-exome sequencing and deep-sequencing data. H.W. helped with data analyses. J.L. supervised the project. Q.L., Y.C., Y.L. and J.L. designed the experiments, analysed the data and wrote the paper. All authors contributed to the manuscript.

Competing interests

The authors declare no competing interests.

Correspondence to Yong Chen or Jinsong Li.

Integrated supplementary information

Supplementary Figure 1 One-step generation of mouse models carrying point mutations via direct injection of BE3 into zygotes.

a, The target sequence in Tyr exon 1. The target sequence is underlined. The PAM sequence and the intended base mutation are shown in blue and red, respectively. b, A mutant mouse (Tyr-7, 2-weeks old, right) produced by intracytoplasmic injection of BE3 mRNA and Tyr sgRNA. c, Genotyping analysis of the mutant mouse in (b) showing a base mutation in Tyr, leading to the replacement of Arginine by a termination codon. d, Progeny with the mutant parents carrying TYRR223stop/R223stop. All 24 pups exhibit an albino phenotype. e, The target sequence in Dnd1 exon 2. The target sequence is underlined. The PAM sequence and the expected base mutation are shown in blue and red, respectively. f, The representative images of WT and mutant (Dnd1-15) gonads carrying an Oct4-EGFP transgene (E12.5). Left, bright-field image of the gonads. Right, the same gonads under fluorescent illumination. Two independent mutants were analyzed. Scale bar, 200 μm. g, Genotyping analysis of the mutant gonad in (f) showing a base mutation in Dnd1, leading to the replacement of Glutamine by a termination codon. h, Summary of base editing efficiency in embryos or mice generated through direct injection of BE3 mRNA and Tyr sgRNA or Dnd1 sgRNA into zygotes. i, An adult mouse (9-weeks old) carrying a point mutation in Dnd1 and its testis. Scale bar, 1.0 mm. j, Histological analysis of the Dnd1 mutant testis shown in i. No germ cells in seminiferous tubules. Scale bar, 100 μm. k, Histological analysis of the ovary from one adult Dnd1 mutant mouse, indicating no germ cells. Scale bar, 100 μm. Two independent mutants were analyzed for each group in i-k.

Supplementary Figure 2 The chromatograms of Sanger sequencing show the homozygous and heterozygous mutations in cell clones and SC embryos from 4B2N1-DP1 cells.

a, b, Sanger sequencing chromatograms of DNA from cell clones (a) and SC embryos (b) obtained from 4B2N1-DP1 cells showing the homozygous (one peak at the target site) and heterozygous (two peaks at the target site) mutations. The nucleotides in red shadow indicate the substituted nucleotide. The PAM sequences are shown in blue shadow and the sgRNA-targeted sequences are underlined. The editing window is shown with dotted orthogon.

Supplementary Figure 3 Analyses of the sgRNA coverage and base-mutation efficiency in cell clones and SC embryos obtained from 4B2N1 cells with an sgRNA library.

a, E12.5 SC embryos from 4B2N1 cells carrying Dnd1 sgRNA library. b, Identification of sgRNA in E12.5 SC embryos by PCR. All tested SC embryos carried sgRNA transgenes. Data represent 3 independent experiments. c, Determination of the inserted sgRNA by Sanger sequencing of RCR products in b. Each sgRNA carried a specific sequence that can be recognized as a ‘barcode’ of the SC embryo. The sgRNA transcription is driven by U6 promoter. d, Top, the number of identified sgRNAs in cell clones and SC embryos from 4B2N1-DP1 cells. Bottom, the number of the sgRNAs that induced homozygous base mutations in cell clones and SC embryos determined by Sanger sequencing. e, Deep-sequencing of sgRNAs in 4B2N1-DP2 cells carrying an sgRNA library consisting of 30 sgRNAs. The horizontal axis represents the sgRNA number. n = 30 sgRNAs. f, Distribution of sgRNAs in 4B2N1-DP2 cells according to their read counts. g, SgRNA distribution in 4B2N1-DP2 cells. Out of 142 cell clones, 76.8%carried only one sgRNA. h, Similar sgRNA distribution patterns revealed by cell-clone and deep-sequencing of 4B2N1-DP2 cells. 109 cell clones with only one sgRNA covering 26 sgRNAs were analyzed. The horizontal axis represents the sgRNA number. The left vertical axis represents the number of cell clones with a specific sgRNA. The right vertical axis represents the frequency of the corresponding sgRNA through deep-sequencing. i, Summary of the mutations in tested cell clones through Sanger sequencing of sgRNA-targeted sites in cell clones with only one sgRNA from 4B2N1-DP2 cells. Other represents the cell clones without sgRNA or with 2 or more sgRNAs. j, The whole-exome sequencing data of three 4B2N1-DP2 cell clones (4B2N1-C1, C2 and C3) from showed a low rate of mosaicism at the target sites of Dnd1. The target sequences are shown with yellow shadow. The PAM sites and substituted nucleotides are shown in green and red, respectively. The expected editing windows are shown with dotted orthogon. The columns on the right indicate frequencies of mutant alleles.

Supplementary Figure 4 Analyses of intended and non-intended base mutations in SC embryos from 4B2N1-DP2 cells and embryos obtained by injection of BE3 into zygotes.

a, SgRNA distribution in a total of 128 E12.5 SC embryos obtained by ICAHCI of 4B2N1-DP2 cells, showing that a majority of the SC embryos (89.8%) carried one sgRNA. b, SgRNA distribution in 115 embryos with one sgRNA, covering a total of 24 different sgRNAs. The horizontal axis represents the sgRNA number. The vertical axis represents the number of SC embryos with a specific sgRNA. c, Summary of the mutations in tested SC embryos with one sgRNA obtained through ICAHCI of 4B2N1-DP2 cells. Other represents the SC embryos without sgRNA or with 2 or more sgRNAs. d, Top, the number of identified sgRNAs in cell clones and SC embryos obtained from 4B2N1-DP2 cells. Bottom, the number of the sgRNAs that induced homozygous base mutations in cell clones and SC embryos determined by Sanger sequencing analyses. e, Blunt Cloning and sequencing analysis of different organs in one SC embryo carrying a heterozygous base mutation (top) and one with a homozygous base mutation (bottom). More than 20 clones were tested for each organ. f, Summary of the generation of E12.5 SC embryos from 4B2N1 cells carrying Dnd1 sgRNA libraries.

Supplementary Figure 5 Functional validation of identified amino acids of DND1 in vivo.

a, Summary of validation experiments. All E12.5 embryos carrying an expected point mutation lost PGCs in gonads. b, Rare PGCs in gonads of E12.5 embryos carrying the V60M (n = 21) or G82R (n= 3) in DND1 obtained by injection of BE3 mRNA and corresponding sgRNA into zygotes (n = the number of mouse for analysis). Scale bar, 200 μm. c, Morphological and histological analyses of the testis and epididymis of the mutant mice. Note no germ cells in the mutant testis and epididymis with V60M or G82R in DND1. Two adult mice (8-weeks old) were used for each group. Scale bar in left column, 1.0 mm; scale bars in middle and right columns, 100 μm. d, The whole-exome sequencing data of four embryos (CI-E1, E2, E3 and E4) generated by injection of BE3 mRNA and corresponding sgRNA into zygotes shows a low rate of mosaicism at the target sites of Dnd1. CI-EC is a wild-type control embryo. The target sequences are shown with yellow shadow. The PAM sites and substituted nucleotides are shown in green and red, respectively. The expected editing windows are shown with dotted orthogon. The columns on the right indicate frequencies of mutant alleles.

Supplementary Figure 6 The E59K, V60M, P76L and G82R of DND1 disrupt the protein stability and protein-protein interaction.

a, Sequence alignment of RRM domains from various proteins. G82 is well conserved in RRM domains. b, In vitro purification of wild-type and mutant RRM domains (1-217) of DND1 in E. coli. c, GST pull-down assay for the interactions of GST-NANOS2 or GST-CNOT1 (1-551) with DND1 and DND1 mutant proteins. d, Transcription analysis of Oct4, Sox2, Nanog, Dnd1, Nanos2 and Cnot1 genes in mouse ESCs (E14). The expression values were normalized to that of Gapdh. Data are shown as the average mean +/− s.d. (n= 3 three independent experiments). The source data can be found in Supplementary Table 13. e, Analysis of expression levels of DND1 and DND1 mutant proteins in ESCs by transfection of HA-tagged Dnd1 plasmids. f, g, Co-immunoprecipitation (Co-IP) analysis in mouse ESCs shows that the mutation of DND1 disrupt the interaction with NANOS2 but did not affect the binding with CNOT1. Data in b, c and e-g represent 3 independent experiments and their unprocessed images of gels are shown in Supplementary Fig. 7.

Supplementary Figure 7

Unprocessed images of key blots/gels in whole study

Supplementary Information

Supplementary Information

Supplementary Figures 1–7 and legends for Supplementary Tables 1–13.

Reporting Summary

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Further reading

Fig. 1: Establishment of an enhanced BE3 system.
Fig. 2: Haploid ESCs carrying a constitutively expressed BE3 with two NLSs (4B2N1 cells) enabling efficient base editing in semi-cloned embryos.
Fig. 3: Base mutation efficiency in 4B2N1 cells carrying an sgRNA library targeting Dnd1.
Fig. 4: Haploid ESCs carrying constitutively expressed BE3 and an sgRNA library support the generation of semi-cloned embryos with base mutations in Dnd1 in one step.
Fig. 5: Functional validation of identified amino acids of DND1 via injection of BE3 mRNA and individual sgRNA into zygotes.
Fig. 6: Structural analysis indicates that the E59K, V60M, P76L and G82R mutations of DND1 are essential for protein function.
Fig. 7: E59K, V60M, P76L and G82R of DND1 deplete PGCs through disruption of protein stability and protein–protein interaction.
Supplementary Figure 1: One-step generation of mouse models carrying point mutations via direct injection of BE3 into zygotes.
Supplementary Figure 2: The chromatograms of Sanger sequencing show the homozygous and heterozygous mutations in cell clones and SC embryos from 4B2N1-DP1 cells.
Supplementary Figure 3: Analyses of the sgRNA coverage and base-mutation efficiency in cell clones and SC embryos obtained from 4B2N1 cells with an sgRNA library.
Supplementary Figure 4: Analyses of intended and non-intended base mutations in SC embryos from 4B2N1-DP2 cells and embryos obtained by injection of BE3 into zygotes.
Supplementary Figure 5: Functional validation of identified amino acids of DND1 in vivo.
Supplementary Figure 6: The E59K, V60M, P76L and G82R of DND1 disrupt the protein stability and protein-protein interaction.
Supplementary Figure 7