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CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes.

Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, Lv J, Xie X, Chen Y, Li Y, Sun Y, Bai Y, Songyang Z, Ma W, Zhou C, Huang J - Protein Cell (2015)

Bottom Line: Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications.We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB).Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.

View Article: PubMed Central - PubMed

Affiliation: Guangdong Province Key Laboratory of Reproductive Medicine, the First Affiliated Hospital, and Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.

ABSTRACT
Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.

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Targeting of theHBBgene in human tripronuclear (3PN) zygotes using CRISPR/Cas9. (A) Four groups of 3PN zygotes were injected intra-cytoplasmically with GFP mRNA (50 ng/μL) and Cas9/gRNA/ssDNA in different concentration combinations. The genomes of GFP+ embryos were first amplified by multiplex displacement amplification. The region spanning the target site was then PCR amplified, subcloned into TA vectors, and sequenced. * Indicates that target fragments in 5 GFP+ embryos failed to be PCR amplified. (B) Sequencing chromatographs of the wild-type allele and recombined allele generated by homologous recombination between HBB and HBD are shown here. The region with base substitution is underlined with red line. (C) A representative sequencing chromatogram of the region spanning the target site in Cas9-cleaved 3PN embryos. Double peaks near the PAM sequence (green) are indicated. (D) Five embryos with double peaks near the PAM sequence were randomly selected for the T7E1 assay. Blue arrowhead indicates the expected size for uncut PCR products. Control, amplified products from target regions with no double peaks near the PAM sequence. (E) Embryo No.16 from group 3 was used to PCR amplify sequences spanning the gRNA target regions of the HBB gene. The PCR products were then subcloned and sequenced. A total of 50 clones were examined, and the number of clones for each pattern indicated. PAM, green. G1 gRNA sequence, blue. Point mutations, red
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Fig2: Targeting of theHBBgene in human tripronuclear (3PN) zygotes using CRISPR/Cas9. (A) Four groups of 3PN zygotes were injected intra-cytoplasmically with GFP mRNA (50 ng/μL) and Cas9/gRNA/ssDNA in different concentration combinations. The genomes of GFP+ embryos were first amplified by multiplex displacement amplification. The region spanning the target site was then PCR amplified, subcloned into TA vectors, and sequenced. * Indicates that target fragments in 5 GFP+ embryos failed to be PCR amplified. (B) Sequencing chromatographs of the wild-type allele and recombined allele generated by homologous recombination between HBB and HBD are shown here. The region with base substitution is underlined with red line. (C) A representative sequencing chromatogram of the region spanning the target site in Cas9-cleaved 3PN embryos. Double peaks near the PAM sequence (green) are indicated. (D) Five embryos with double peaks near the PAM sequence were randomly selected for the T7E1 assay. Blue arrowhead indicates the expected size for uncut PCR products. Control, amplified products from target regions with no double peaks near the PAM sequence. (E) Embryo No.16 from group 3 was used to PCR amplify sequences spanning the gRNA target regions of the HBB gene. The PCR products were then subcloned and sequenced. A total of 50 clones were examined, and the number of clones for each pattern indicated. PAM, green. G1 gRNA sequence, blue. Point mutations, red

Mentions: To investigate the specificity and efficacy of gene targeting in human tripronuclear (3PN) zygotes, we co-injected G1 gRNA, Cas9 mRNA, GFP mRNA, and the ssDNA oligo into the cytoplasm of human 3PN zygotes in different concentration combinations (Fig. 2A). Based on morphology, ~80% of the embryos remained viable 48 h after injection (Fig. 2A), in agreement with low toxicity of Cas9 injection in mouse embryos (Wang et al., 2013; Yang et al., 2013). All GFP-positive embryos were then collected for whole-genome amplification by multiplex displacement amplification (Dean et al., 2002; Hosono et al., 2003), followed by PCR amplification of the G1 gRNA target region and sequencing. Of the 54 PCR-amplified embryos, 28 were cleaved by Cas9, indicating an efficiency of ~52% (Fig. 2A). Furthermore, 4 of the 28 Cas9-cleaved embryos (14.3%) were clearly edited using the ssDNA oligo as a repair template (Fig. 2A). Additionally, 7 embryos contained four identical point mutations in tandem, an clear indication of HDR using the HBD gene as a repair template (Fig. 2A and 2B). This finding suggests recombination of the HBB gene with HBD in 7 out of the 28 cleaved embryos (25%), even in the presence of co-injected exogenous ssDNA donor template (Fig. 2A and 2B). Similar observations have been found in mouse embryos, where endogenous homologous templates were found to compete with ssDNA oligos for HDR repair (Wu et al., 2013).Figure 2


CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes.

Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, Lv J, Xie X, Chen Y, Li Y, Sun Y, Bai Y, Songyang Z, Ma W, Zhou C, Huang J - Protein Cell (2015)

Targeting of theHBBgene in human tripronuclear (3PN) zygotes using CRISPR/Cas9. (A) Four groups of 3PN zygotes were injected intra-cytoplasmically with GFP mRNA (50 ng/μL) and Cas9/gRNA/ssDNA in different concentration combinations. The genomes of GFP+ embryos were first amplified by multiplex displacement amplification. The region spanning the target site was then PCR amplified, subcloned into TA vectors, and sequenced. * Indicates that target fragments in 5 GFP+ embryos failed to be PCR amplified. (B) Sequencing chromatographs of the wild-type allele and recombined allele generated by homologous recombination between HBB and HBD are shown here. The region with base substitution is underlined with red line. (C) A representative sequencing chromatogram of the region spanning the target site in Cas9-cleaved 3PN embryos. Double peaks near the PAM sequence (green) are indicated. (D) Five embryos with double peaks near the PAM sequence were randomly selected for the T7E1 assay. Blue arrowhead indicates the expected size for uncut PCR products. Control, amplified products from target regions with no double peaks near the PAM sequence. (E) Embryo No.16 from group 3 was used to PCR amplify sequences spanning the gRNA target regions of the HBB gene. The PCR products were then subcloned and sequenced. A total of 50 clones were examined, and the number of clones for each pattern indicated. PAM, green. G1 gRNA sequence, blue. Point mutations, red
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4417674&req=5

Fig2: Targeting of theHBBgene in human tripronuclear (3PN) zygotes using CRISPR/Cas9. (A) Four groups of 3PN zygotes were injected intra-cytoplasmically with GFP mRNA (50 ng/μL) and Cas9/gRNA/ssDNA in different concentration combinations. The genomes of GFP+ embryos were first amplified by multiplex displacement amplification. The region spanning the target site was then PCR amplified, subcloned into TA vectors, and sequenced. * Indicates that target fragments in 5 GFP+ embryos failed to be PCR amplified. (B) Sequencing chromatographs of the wild-type allele and recombined allele generated by homologous recombination between HBB and HBD are shown here. The region with base substitution is underlined with red line. (C) A representative sequencing chromatogram of the region spanning the target site in Cas9-cleaved 3PN embryos. Double peaks near the PAM sequence (green) are indicated. (D) Five embryos with double peaks near the PAM sequence were randomly selected for the T7E1 assay. Blue arrowhead indicates the expected size for uncut PCR products. Control, amplified products from target regions with no double peaks near the PAM sequence. (E) Embryo No.16 from group 3 was used to PCR amplify sequences spanning the gRNA target regions of the HBB gene. The PCR products were then subcloned and sequenced. A total of 50 clones were examined, and the number of clones for each pattern indicated. PAM, green. G1 gRNA sequence, blue. Point mutations, red
Mentions: To investigate the specificity and efficacy of gene targeting in human tripronuclear (3PN) zygotes, we co-injected G1 gRNA, Cas9 mRNA, GFP mRNA, and the ssDNA oligo into the cytoplasm of human 3PN zygotes in different concentration combinations (Fig. 2A). Based on morphology, ~80% of the embryos remained viable 48 h after injection (Fig. 2A), in agreement with low toxicity of Cas9 injection in mouse embryos (Wang et al., 2013; Yang et al., 2013). All GFP-positive embryos were then collected for whole-genome amplification by multiplex displacement amplification (Dean et al., 2002; Hosono et al., 2003), followed by PCR amplification of the G1 gRNA target region and sequencing. Of the 54 PCR-amplified embryos, 28 were cleaved by Cas9, indicating an efficiency of ~52% (Fig. 2A). Furthermore, 4 of the 28 Cas9-cleaved embryos (14.3%) were clearly edited using the ssDNA oligo as a repair template (Fig. 2A). Additionally, 7 embryos contained four identical point mutations in tandem, an clear indication of HDR using the HBD gene as a repair template (Fig. 2A and 2B). This finding suggests recombination of the HBB gene with HBD in 7 out of the 28 cleaved embryos (25%), even in the presence of co-injected exogenous ssDNA donor template (Fig. 2A and 2B). Similar observations have been found in mouse embryos, where endogenous homologous templates were found to compete with ssDNA oligos for HDR repair (Wu et al., 2013).Figure 2

Bottom Line: Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications.We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB).Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.

View Article: PubMed Central - PubMed

Affiliation: Guangdong Province Key Laboratory of Reproductive Medicine, the First Affiliated Hospital, and Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.

ABSTRACT
Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.

Show MeSH