CRISPR/Cas9-mediated editing of HBB gene in human cells

The human β-globin (HBB) gene, which encodes a subunit of the adult hemoglobin and is mutated in β-thalassemia (Hill et al., 1962). In China, CD14/15, CD17, and CD41/42, which are frame-shift or truncated mutations of β-globin, are three of the most common β-thalassemia mutations (Cao and Galanello, 2010). Located on chromosome 11, HBB is within the β-globin gene cluster that contains four other globin genes with the order of (from 5′ to 3′) HBE, HBG2, HBG1, HBD, and HBB (Schechter, 2008). Because the sequences of HBB and HBD are very similar, HBD may also be used as a template to repair HBB. The HBD footprints left in the repaired HBB locus should enable us to investigate whether and how endogenous homologous sequences may be utilized as HDR templates, information that will prove invaluable to any future endeavors that may employ CRISPR/Cas9 to repair gene loci with repeated sequences.

Using online tools developed by Feng Zhang and colleagues (http://crispr.mit.edu/), we designed and generated three gRNAs (named G1, G2, and G3) that targeted different regions of the HBB gene (Fig. 1A), and transfected the gRNA-Cas9 expression vectors into human 293T cells. Compared with the GFP mock vector, G1 and G2 gRNAs exhibited efficient cleavage activities as determined by the T7E1 assay (Fig. 1B) (Shen et al., 2014). Sequencing analysis of the two regions targeted by G1 and G2 revealed distinct indel spectra, reflecting different NHEJ repair preferences at these two sites (Fig. S1). CRISPR/Cas9 targeting of the β-globin locus was previously reported to have substantially high off-target activity in cultured human cells (Cradick et al., 2013). We therefore designed specific PCR primers for the top 7 predicted off-target sites in the genome for both G1 and G2 gRNAs, along with the predicted off-target site of G1 gRNA in the HBD gene (Table S1). We then carried out the T7E1 assay to assess the off-target effects of G1 and G2 gRNAs in human 293T cells. While G2 gRNA showed very low off-target cleavage activity in the intergenic region (G2-OT4) (Fig. S2), gRNA G1 did not exhibit detectable off-target cleavage at the top 7 predicted off-target sites (Fig. 1C). Furthermore, we also failed to find sequence modifications at the predicted site in the HBD gene, despite close sequence similarity between HBD and HBB (Fig. 1D). These data suggest that the G1 gRNA to be a better candidate for further studies. Next, we synthesized a ssDNA oligo donor template that encoded 6 silent mutations and transfected this oligo alone or together with the G1 gRNA-Cas9 plasmid into 293T cells (Fig. 1E). We then extracted genomic DNA from the cells 48 h later for PCR amplification of the G1 target region. The PCR products were subsequently subcloned for sequencing. Compared to none from oligo-only control, analysis of 29 independent clones revealed 14 clones (48.3%) that perfectly matched the donor oligo template (Fig. 1E), indicating high efficiency of our approach and precise editing of the HBB locus in cells.

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) Full size image

CRISPR/Cas9-mediated editing of HBB gene in human tripronuclear zygotes

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 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 Full size image

Because of the preference for the error-prone NHEJ pathway, the HBB sequences from Cas9-cleaved embryos showed double peaks near the PAM site on sequencing chromatographs (Fig. 2C). Analysis of 5 of these embryos using the T7E1 assay also confirmed successful cleavage by G1 gRNA and Cas9 (Fig. 2D). In addition, the gene-edited embryos were mosaic. For example, embryo No. 16 contained many different kinds of alleles (Fig. 2E).

CRISPR/Cas9 has off-target effect in human tripronuclear embryos

To determine the off-target effects of CRISPR/Cas9 in these embryos, we again examined the top 7 potential off-target sites plus the site in the HBD gene. The T7E1 assay revealed off-target cleavage in the OPCML intron (G1-OT4) and the TULP1 intron (G1-OT5) (Figs. 3A, S3 and S4), although none of these sites appeared to be cleaved in human 293T cells (Fig. 1C). We then randomly selected 6 HBB-cleaved embryos (three each from groups 2 and 3, Fig. 2A) for whole-exome sequencing. As shown in Fig. 3B, on-target indels were identified in all of the samples. Two candidate off-target sites within exons were found, where lower concentration of the Cas9 mRNA and gRNA had been used (sample A and C, Fig. 3B), and further confirmed through the T7E1 assay (Fig. S5). These two sites reside in the exons of the C1QC and Transthyretin (TTR) gene, both of which closely match the G1 gRNA sequence in the seed region (Fig. S6). These data demonstrate that CRISPR/Cas9 has notable off-target effects in human 3PN embryos.

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 Full size image

HDR of double strand breaks at the HBB gene occurs preferentially through the non-crossover pathway

DSBs can be repaired through either error-prone NHEJ or high-fidelity HDR (Ciccia and Elledge, 2010; Moynahan and Jasin, 2010). There are three options for the HDR pathway, non-crossover synthesis-dependent strand annealing (SDSA), non-crossover double-strand break repair (DSBR), and crossover DSBR (Fig. 4A). Bi-directional sequence exchange between the recombined genes occurs with crossover, while uni-directional sequence exchange occurs in absence of crossover. Of the 3PN embryos examined thus far, 4 were repaired using the ssDNA oligo as template and 7 were recombined with the endogenous HBD gene (Fig. 2A). When the HBD locus from the 7 recombined 3PN embryos were amplified and examined, we found that the HBD locus in the 5 successfully-amplified embryos remained intact, containing no HBB sequences (Fig. 4B). This lack of bi-directional sequence exchange supports the notion that the HBB gene was repaired primarily through non-crossover HDR (San Filippo et al., 2008). It is possible that one of the alleles in 3PN embryo No.16 (group 3) (Fig. 2E), which only contained 4 of the 6 silent mutations from the ssDNA oligo, might have been generated by non-crossover pathway as well (Fig. 4A). Taken together, our results suggest that homologous recombination in human early embryos preferentially occur through the non-crossover HDR pathway (Fig. 4C), similar to what has been observed in human iPS cells (Byrne et al., 2014).