An oocyte-specific Cas9-expressing mouse for germline CRISPR/Cas9-mediated genome editing

Abstract

Cas9 transgenes can be employed for genome editing in mouse zygotes. However, using transgenic instead of exogenous Cas9 to produce gene-edited animals creates unique issues including ill-defined transgene integration sites, the potential for prolonged Cas9 expression in transgenic embryos, and increased genotyping burden. To overcome these issues, we generated mice harboring an oocyte-specific, Gdf9 promoter driven, Cas9 transgene (Gdf9-Cas9) targeted as a single copy into the Hprt1 locus. The X-linked Hprt1 locus was selected because it is a defined integration site that does not influence transgene expression, and breeding of transgenic males generates obligate transgenic females to serve as embryo donors. Using microinjections and electroporation to introduce sgRNAs into zygotes derived from transgenic dams, we demonstrate that Gdf9-Cas9 mediates genome editing as efficiently as exogenous Cas9 at several loci. We show that genome editing efficiency is independent of transgene inheritance, verifying that maternally derived Cas9 facilitates genome editing. We also show that paternal inheritance of Gdf9-Cas9 does not mediate genome editing, confirming that Gdf9-Cas9 is not expressed in embryos. Finally, we demonstrate that off-target mutagenesis is equally rare when using transgenic or exogenous Cas9. Together, these results show that the Gdf9-Cas9 transgene is a viable alternative to exogenous Cas9.

Keywords: CRISPR; SpCas9; genome editing; germline mutations.

Figure 1.

Targeting Gdf9-Cas9 to the Hprt1 locus through a complementation design in MESC. (A) Schematic representing the endogenous deletion locus at Hprt1^b-m3 (not drawn to scale). The complementation targeting construct contains Gdf9 promoter sequence, the open reading frame of S. pyogenes Cas9 (SpCas9), promoter and exon 1 sequence from human HPRT1 (shown in red), and intron 1, exon 2, and intron 2 sequence from mouse Hprt1. Homologous recombination introduces the Gdf9-SpCas9 transgene into the genome and restores Hprt1 function. (B) After culturing the electroporated ES cells in Hypoxanthine-Aminopterin-Thymidine (HAT) medium and picking clones, additional PCR-based screening was completed to identify correctly targeted clones. (1) Primers span the 36 kb deletion (Hprt1 b-m3, primers a/b in A) identified clones with admixtures of non-targeted ES cells; only cells still harboring the 36 kb deletion would produce the 380 bp product (pink clone IDs) (2) Positive control PCR to generate a product from the human HPRT1 complementation with successful targeting (HPRT1-CS, primers c/d in A; all clones should have had the 450 bp band) (3) Internal control PCR for Cas9 open reading frame (ORF; 300 bp product, primers e/f in A); clones that were selected for blastocyst injections have green identifiers. (C) Sanger sequencing of a portion of the Gdf9 promoter and the Cas9 ORF confirmed allele integrity following germline transmission from chimeras established from targeted ES cells. Sequencing reads are shown numerically with arrows indicating the orientation of sequencing (1-11).

Figure 1.

Targeting Gdf9-Cas9 to the Hprt1 locus through a complementation design in MESC. (A) Schematic representing the endogenous deletion locus at Hprt1^b-m3 (not drawn to scale). The complementation targeting construct contains Gdf9 promoter sequence, the open reading frame of S. pyogenes Cas9 (SpCas9), promoter and exon 1 sequence from human HPRT1 (shown in red), and intron 1, exon 2, and intron 2 sequence from mouse Hprt1. Homologous recombination introduces the Gdf9-SpCas9 transgene into the genome and restores Hprt1 function. (B) After culturing the electroporated ES cells in Hypoxanthine-Aminopterin-Thymidine (HAT) medium and picking clones, additional PCR-based screening was completed to identify correctly targeted clones. (1) Primers span the 36 kb deletion (Hprt1 b-m3, primers a/b in A) identified clones with admixtures of non-targeted ES cells; only cells still harboring the 36 kb deletion would produce the 380 bp product (pink clone IDs) (2) Positive control PCR to generate a product from the human HPRT1 complementation with successful targeting (HPRT1-CS, primers c/d in A; all clones should have had the 450 bp band) (3) Internal control PCR for Cas9 open reading frame (ORF; 300 bp product, primers e/f in A); clones that were selected for blastocyst injections have green identifiers. (C) Sanger sequencing of a portion of the Gdf9 promoter and the Cas9 ORF confirmed allele integrity following germline transmission from chimeras established from targeted ES cells. Sequencing reads are shown numerically with arrows indicating the orientation of sequencing (1-11).

Figure 1.

Targeting Gdf9-Cas9 to the Hprt1 locus through a complementation design in MESC. (A) Schematic representing the endogenous deletion locus at Hprt1^b-m3 (not drawn to scale). The complementation targeting construct contains Gdf9 promoter sequence, the open reading frame of S. pyogenes Cas9 (SpCas9), promoter and exon 1 sequence from human HPRT1 (shown in red), and intron 1, exon 2, and intron 2 sequence from mouse Hprt1. Homologous recombination introduces the Gdf9-SpCas9 transgene into the genome and restores Hprt1 function. (B) After culturing the electroporated ES cells in Hypoxanthine-Aminopterin-Thymidine (HAT) medium and picking clones, additional PCR-based screening was completed to identify correctly targeted clones. (1) Primers span the 36 kb deletion (Hprt1 b-m3, primers a/b in A) identified clones with admixtures of non-targeted ES cells; only cells still harboring the 36 kb deletion would produce the 380 bp product (pink clone IDs) (2) Positive control PCR to generate a product from the human HPRT1 complementation with successful targeting (HPRT1-CS, primers c/d in A; all clones should have had the 450 bp band) (3) Internal control PCR for Cas9 open reading frame (ORF; 300 bp product, primers e/f in A); clones that were selected for blastocyst injections have green identifiers. (C) Sanger sequencing of a portion of the Gdf9 promoter and the Cas9 ORF confirmed allele integrity following germline transmission from chimeras established from targeted ES cells. Sequencing reads are shown numerically with arrows indicating the orientation of sequencing (1-11).

Figure 2.

Breeding scheme to generate zygotes for genome editing and to achieve germline transmission. Male carriers of the Hprt1^Gdf9-Cas9 transgene, delineated in orange, are bred with wild-type females to give rise to obligate Hprt1^Gdf9-Cas9/+ daughters. The obligate females thereby do not need to be genotyped prior to superovulation, saving time when optimal age for superovulation is at 4 weeks for C57BL/6NJ mice. The obligate Hprt1^Gdf9-Cas9/+ females are mated with wild-type males to generate both male and female zygotes, with and without inheritance of Hprt1^Gdf9-Cas9. Zygotes are collected at E0.5, when Cas9 is expressed (delineated in green), and manipulated with genome editing reagents to either (1) culture in vitro for 4 days to generate blastocysts for subsequent genotyping or (2) transfer to pseudopregnant females to produce live-born founder (F0) animals, which are screened for the desired mutation. Male F0s harboring a desired gene edit are preferentially bred over targeted female F0s. The resulting offspring are screened for the on-target mutation. Targeted male progeny from F0 founder sires are preferentially selected as they cannot inherit the Hprt1^Gdf9-Cas9 allele from a male F0.

Figure 3.

Expression of Cas9 co-localizes with Gdf9 in oocytes. Hprt1^Gdf9-Cas9/+, Rosa26^CAG-Cas9 (ubiquitous Cas9 expression, positive control) and wild-type (Gdf9 expression positive control; Cas9 negative control) females were sacrificed for tissue collection between postnatal day (P)21-25. Tissues were fresh frozen for cryosectioning and RNA in situ hybridization. Probes were generated to hybridize to Gdf9 and Cas9.

Figure 4.

Genome editing is dependent upon maternally supplied Cas9. (A) Male Hprt1^Gdf9-Cas9/Y mice were mated to superovulated wild-type C57BL/6N females to generate obligate Hprt1^Gdf9-Cas9 female zygotes. Two gRNAs targeting Nanos2 for an interval deletion were electroporated into zygotes and embryos were transferred to pseudopregnant females. (B) Male mice were identified by Sry genotyping and were excluded from analysis. Female mice were genotyped for both Hprt1^Gdf9-Cas9 and the Nanos2 interval deletion. PCR genotyping demonstrates that all females were WT for the Nanos2 deletion allele (WT product 629 bp, targeted ~275 bp) and carriers of the Hprt1^Gdf9-Cas9 allele. The positive control (+) for the Nanos2 deletion allele was obtained from Cas9 targeting by microinjection. A separate sample was used as a positive control for the Hprt1^Gdf9-Cas9 transgene. Wild-type mice were used as negative controls.

Figure 4.

Genome editing is dependent upon maternally supplied Cas9. (A) Male Hprt1^Gdf9-Cas9/Y mice were mated to superovulated wild-type C57BL/6N females to generate obligate Hprt1^Gdf9-Cas9 female zygotes. Two gRNAs targeting Nanos2 for an interval deletion were electroporated into zygotes and embryos were transferred to pseudopregnant females. (B) Male mice were identified by Sry genotyping and were excluded from analysis. Female mice were genotyped for both Hprt1^Gdf9-Cas9 and the Nanos2 interval deletion. PCR genotyping demonstrates that all females were WT for the Nanos2 deletion allele (WT product 629 bp, targeted ~275 bp) and carriers of the Hprt1^Gdf9-Cas9 allele. The positive control (+) for the Nanos2 deletion allele was obtained from Cas9 targeting by microinjection. A separate sample was used as a positive control for the Hprt1^Gdf9-Cas9 transgene. Wild-type mice were used as negative controls.

Figure 5.

Off-target genome editing occurs with both endogenous and exogenous Cas9. Zygotes were generated from wild-type and Hprt1^Gdf9-Cas9 females and microinjected or electroporated with or without exogenous Cas9, respectively, and two gRNAs targeting Tyr (N=8 animals or blastocysts per Cas9 source/route of administration). Sanger sequencing and ICE analysis of sequencing traces were performed to detect genome editing at the on-target and top 4 predicted off-target sites for each gRNA and to calculate indel allele contribution. (A) Scatter plot of ICE score for each Cas9 source/route of administration for the Tyr on-target site. Endogenous MI and EP samples with inheritance of the Hprt1^Gdf9-Cas9 transgene are displayed as triangles, all other samples are circles. (B) Scatter plot of ICE score for each Cas9 source/route of administration for one off-target site. Of the 8 off-target loci screened for the 2 gRNAs employed (4 per guide), mutagenesis was detected at only this off-target site. Of the 2 mice with an off-target event from the Endogenous MI samples, one had inheritance of the Hprt1^Gdf9-Cas9 transgene (triangle). (C) Scatter plot depicting off-target ICE score versus on-target score for the only off-target site with identified mutagenesis events to illustrate occurrence of on- and off-target events occurring within the same sample. Endo, endogenous Cas9; Exo, exogenous Cas9; MI, microinjection; EP, electroporation; (T), Hprt1^Gdf9-Cas9 transgenic.

Figure 5.

Off-target genome editing occurs with both endogenous and exogenous Cas9. Zygotes were generated from wild-type and Hprt1^Gdf9-Cas9 females and microinjected or electroporated with or without exogenous Cas9, respectively, and two gRNAs targeting Tyr (N=8 animals or blastocysts per Cas9 source/route of administration). Sanger sequencing and ICE analysis of sequencing traces were performed to detect genome editing at the on-target and top 4 predicted off-target sites for each gRNA and to calculate indel allele contribution. (A) Scatter plot of ICE score for each Cas9 source/route of administration for the Tyr on-target site. Endogenous MI and EP samples with inheritance of the Hprt1^Gdf9-Cas9 transgene are displayed as triangles, all other samples are circles. (B) Scatter plot of ICE score for each Cas9 source/route of administration for one off-target site. Of the 8 off-target loci screened for the 2 gRNAs employed (4 per guide), mutagenesis was detected at only this off-target site. Of the 2 mice with an off-target event from the Endogenous MI samples, one had inheritance of the Hprt1^Gdf9-Cas9 transgene (triangle). (C) Scatter plot depicting off-target ICE score versus on-target score for the only off-target site with identified mutagenesis events to illustrate occurrence of on- and off-target events occurring within the same sample. Endo, endogenous Cas9; Exo, exogenous Cas9; MI, microinjection; EP, electroporation; (T), Hprt1^Gdf9-Cas9 transgenic.

Figure 5.

Off-target genome editing occurs with both endogenous and exogenous Cas9. Zygotes were generated from wild-type and Hprt1^Gdf9-Cas9 females and microinjected or electroporated with or without exogenous Cas9, respectively, and two gRNAs targeting Tyr (N=8 animals or blastocysts per Cas9 source/route of administration). Sanger sequencing and ICE analysis of sequencing traces were performed to detect genome editing at the on-target and top 4 predicted off-target sites for each gRNA and to calculate indel allele contribution. (A) Scatter plot of ICE score for each Cas9 source/route of administration for the Tyr on-target site. Endogenous MI and EP samples with inheritance of the Hprt1^Gdf9-Cas9 transgene are displayed as triangles, all other samples are circles. (B) Scatter plot of ICE score for each Cas9 source/route of administration for one off-target site. Of the 8 off-target loci screened for the 2 gRNAs employed (4 per guide), mutagenesis was detected at only this off-target site. Of the 2 mice with an off-target event from the Endogenous MI samples, one had inheritance of the Hprt1^Gdf9-Cas9 transgene (triangle). (C) Scatter plot depicting off-target ICE score versus on-target score for the only off-target site with identified mutagenesis events to illustrate occurrence of on- and off-target events occurring within the same sample. Endo, endogenous Cas9; Exo, exogenous Cas9; MI, microinjection; EP, electroporation; (T), Hprt1^Gdf9-Cas9 transgenic.