CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes - PubMed (original) (raw)
doi: 10.1007/s13238-015-0153-5. Epub 2015 Apr 18.
Yanwen Xu # 1, Xiya Zhang # 1, Chenhui Ding # 1, Rui Huang 1, Zhen Zhang 1, Jie Lv 1, Xiaowei Xie 1, Yuxi Chen 1, Yujing Li 1, Ying Sun 1, Yaofu Bai 1, Zhou Songyang 1, Wenbin Ma 1, Canquan Zhou 1, Junjiu Huang 1
Affiliations
- PMID: 25894090
- PMCID: PMC4417674
- DOI: 10.1007/s13238-015-0153-5
CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes
Puping Liang et al. Protein Cell. 2015 May.
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.
Figures
Figure 1
Targeting of the HBB gene in human cells using CRISPR/Cas9. (A) Three gRNA targeting sites were selected for the HBB locus, and the sequence for each gRNA (G1, G2, and G3) is shown with the PAM sequence in green. The three common HBB mutations found in β-thalassemia are indicated in red. Exons are represented by deep blue boxes with yellow arrows indicating transcriptional direction. (B) 293T cells were individually transfected with the three gRNA-Cas9 expression vectors and harvested for genomic DNA isolation 48 h after transfection. A GFP expression vector was used as transfection control. The regions spanning the gRNA target sites were then PCR amplified for the T7E1 assay. Blue arrowhead indicates the expected size for uncut (no mismatch) PCR products. (C) 293T cells were transfected with increasing concentrations (1 μg, 2 μg, 3 μg, 4 μg) of the G1 gRNA-Cas9 vector. A GFP expression vector was used as transfection control. Regions spanning the top 7 predicted off-target sites for each gRNA were PCR amplified for the T7E1 assay. OT, off-target. HBB, on-target editing in the HBB gene locus. (D) The region within the HBD locus that is highly similar to the G1 gRNA-Cas9 target sequence was analyzed as in (C). (E) A ssDNA oligo (Oligo donor) encoding 6 silent mutations (indicated in red) was synthesized (top), and co-transfected with the G1 gRNA-Cas9 construct (pX330-G1) into 293T cells (middle). At 48 h after transfection, genomic DNA was extracted to PCR amplify the region spanning the G1 target site. The PCR products were then subcloned into TA cloning vectors for sequencing analysis. Representative sequencing chromatographs for wild-type and edited alleles are shown with the mutated target region underlined in red (bottom)
Figure 2
Targeting of the HBB gene 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
Figure 3
Off-target cleavage of CRISPR/Cas9 in human 3PN embryos. (A) Off-target cleavage in human embryos was summarized here. PAM sequence are labeled in green. HBB, on-target cleavage of the HBB locus. OT1–7, the top 7 predicted off-target sites. HBD, the predicted off-target site in the HBD locus. Mismatched nucleotides compared to the HBB locus are labeled in red. Some of the off-target sites failed to be amplified by PCR in this experiment. (B) Six Cas9-cleaved embryos were randomly selected (three each from groups 2 and 3) for whole-exome sequencing. Concentrations of the Cas9/gRNAs used for injections are indicated. Candidate off-target sites were also confirmed by T7E1 assay
Figure 4
Repair of double-strand breaks at the HBB gene in human early embryos occurs preferentially through the non-crossover pathway when HDR is utilized. (A) In human cells, DSBs may be repaired through the double-strand break repair (DSBR) pathway or the non-crossover synthesis-dependent strand annealing (SDSA) pathway. Both crossover and non-crossover DSBR can occur. (B) The HBD locus from the 7 recombined 3PN embryos were similarly examined as above. * Indicates that the HBD locus failed to be amplified in two of the embryos. (C) In human embryos, repair of DSBs generated by CRISPR/Cas9 occurs mainly through NHEJ. If HDR is utilized, the non-crossover pathway is preferred
Comment in
- The Genie Is Out of the Bottle.
Brenan CJ. Brenan CJ. IEEE Pulse. 2015 Jul-Aug;6(4):3. doi: 10.1109/mpul.2015.2432991. IEEE Pulse. 2015. PMID: 26394455 No abstract available. - Gene Editing and Germ-line Intervention: The Need for Novel Responses to Novel Technologies.
Addison C, Taylor-Alexander S. Addison C, et al. Mol Ther. 2015 Nov;23(11):1678-1680. doi: 10.1038/mt.2015.185. Mol Ther. 2015. PMID: 26606661 Free PMC article. No abstract available. - UK bioethicists eye designer babies and CRISPR cows.
Ledford H. Ledford H. Nature. 2016 Oct 6;538(7623):17. doi: 10.1038/nature.2016.20713. Nature. 2016. PMID: 27708321 No abstract available.
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References
- Balakier H (1993) Tripronuclear human zygotes: the first cell cycle and subsequent development. Hum Reprod 8:1892–1897 - PubMed
- Bredenoord AL, Pennings G, de Wert G (2008) Ooplasmic and nuclear transfer to prevent mitochondrial DNA disorders: conceptual and normative issues. Hum Reprod Update 14:669–678 - PubMed
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