Xiya Zhang

CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes

ABSTRACTGenome 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.

The Daxx/Atrx Complex Protects Tandem Repetitive Elements during DNA Hypomethylation by Promoting H3K9 Trimethylation.

In mammals, DNA methylation is essential for protecting repetitive sequences from aberrant transcription and recombination. In some developmental contexts (e.g., preimplantation embryos) DNA is hypomethylated but repetitive elements are not dysregulated, suggesting that alternative protection mechanisms exist. Here we explore the processes involved by investigating the role of the chromatin factors Daxx and Atrx. Using genome-wide binding and transcriptome analysis, we found that Daxx and Atrx have distinct chromatin-binding profiles and are co-enriched at tandem repetitive elements in wild-type mouse ESCs. Global DNA hypomethylation further promoted recruitment of the Daxx/Atrx complex to tandem repeat sequences, including retrotransposons and telomeres. Knockdown of Daxx/Atrx in cells with hypomethylated genomes exacerbated aberrant transcriptional de-repression of repeat elements and telomere dysfunction. Mechanistically, Daxx/Atrx-mediated repression seems to involve Suv39h recruitment and H3K9 trimethylation. Our data therefore suggest that Daxx and Atrx safeguard the genome by silencing repetitive elements when DNA methylation levels are low.

Correction of β-thalassemia mutant by base editor in human embryos

Abstractβ-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB −28 (A>G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB −28 (A>G) mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB −28 (A>G) homozygous mutation. Data showed that base editor could precisely correct HBB −28 (A>G) mutation in the patient’s primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM) oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB −28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.

Effective gene editing by high-fidelity base editor 2 in mouse zygotes

ABSTRACTTargeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease-causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical application of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high-fidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.

Efficient Production of Gene-Modified Mice using Staphylococcus aureus Cas9

The CRISPR/Cas system is an efficient genome-editing tool to modify genes in mouse zygotes. However, only the Streptococcus pyogenes Cas9 (SpCas9) has been systematically tested for generating gene-modified mice. The protospacer adjacent motif (PAM, 5′-NGG-3′) recognized by SpCas9 limits the number of potential target sites for this system. Staphylococcus aureus Cas9 (SaCas9), with its smaller size and unique PAM (5′-NNGRRT-3′) preferences, presents an alternative for genome editing in zygotes. Here, we showed that SaCas9 could efficiently and specifically edit the X-linked gene Slx2 and the autosomal gene Zp1 in mouse zygotes. SaCas9-mediated disruption of the tyrosinase (Tyr) gene led to C57BL/6J mice with mosaic coat color. Furthermore, multiplex targeting proved efficient multiple genes disruption when we co-injected gRNAs targeting Slx2, Zp1, and Tyr together with SaCas9 mRNA. We were also able to insert a Flag tag at the C-terminus of histone H1c, when a Flag-encoding single-stranded DNA oligo was co-introduced into mouse zygotes with SaCas9 mRNA and the gRNA. These results indicate that SaCas9 can specifically cleave the target gene locus, leading to successful gene knock-out and precise knock-in in mouse zygotes, and highlight the potential of using SaCas9 for genome editing in preimplantation embryos and producing gene-modified animal models.

CRISPR/Cas9 Promotes Functional Study of Testis Specific X-Linked Gene In Vivo.

Mammalian spermatogenesis is a highly regulated multistage process of sperm generation. It is hard to uncover the real function of a testis specific gene in vitro since the in vitro model is not yet mature. With the development of the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9) system, we can now rapidly generate knockout mouse models of testis specific genes to study the process of spermatogenesis in vivo. SYCP3-like X-linked 2 (SLX2) is a germ cell specific component, which contains a Cor1 domain and belongs to the XLR (X-linked, lymphocyte regulated) family. Previous studies suggested that SLX2 might play an important role in mouse spermatogenesis based on its subcellular localization and interacting proteins. However, the function of SLX2 in vivo is still elusive. Here, to investigate the functions of SLX2 in spermatogenesis, we disrupted the Slx2 gene by using the CRISPR/Cas9 system. Since Slx2 is a testis specific X-linked gene, we obtained knockout male mice in the first generation and accelerated the study process. Compared with wild-type mice, Slx2 knockout mice have normal testis and epididymis. Histological observation of testes sections showed that Slx2 knockout affected none of the three main stages of spermatogenesis: mitosis, meiosis and spermiogenesis. In addition, we further confirmed that disruption of Slx2 did not affect the number of spermatogonial stem cells, meiosis progression or XY body formation by immunofluorescence analysis. As spermatogenesis was normal in Slx2 knockout mice, these mice were fertile. Taken together, we showed that Slx2 itself is not an essential gene for mouse spermatogenesis and CRISPR/Cas9 technique could speed up the functional study of testis specific X-linked gene in vivo.

Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) promotes non-homologous end joining and inhibits homologous recombination repair upon DNA damage

Background: The role of SMCHD1 in DNA damage response is largely unknown. Results: SMCHD1 recruitment to DNA damage foci is regulated by 53BP1. Knocking out SMCHD1 compromised cell survival, and decreased the efficiency of non-homologous end joining (NHEJ) while elevating the efficiency of homologous recombination (HR). Conclusion: SMCHD1 regulates both NHEJ and HR. Significance: Our findings should further understanding of how cells adopt different repair pathways. Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) has been shown to be involved in gene silencing and DNA damage. However, the exact mechanisms of how SMCHD1 participates in DNA damage remains largely unknown. Here we present evidence that SMCHD1 recruitment to DNA damage foci is regulated by 53BP1. Knocking out SMCHD1 led to aberrant γH2AX foci accumulation and compromised cell survival upon DNA damage, demonstrating the critical role of SMCHD1 in DNA damage repair. Following DNA damage induction, SMCHD1 depletion resulted in reduced 53BP1 foci and increased BRCA1 foci, as well as less efficient non-homologous end joining (NHEJ) and elevated levels of homologous recombination (HR). Taken together, these results suggest an important function of SMCHD1 in promoting NHEJ and repressing HR repair in response to DNA damage.

Developmental history and application of CRISPR in human disease

Genome‐editing tools are programmable artificial nucleases, mainly including zinc‐finger nucleases, transcription activator‐like effector nucleases and clustered regularly interspaced short palindromic repeat (CRISPR). By recognizing and cleaving specific DNA sequences, genome‐editing tools make it possible to generate site‐specific DNA double‐strand breaks (DSBs) in the genome. DSBs will then be repaired by either error‐prone nonhomologous end joining or high‐fidelity homologous recombination mechanisms. Through these two different mechanisms, endogenous genes can be knocked out or precisely repaired/modified. Rapid developments in genome‐editing tools, especially CRISPR, have revolutionized human disease models generation, for example, various zebrafish, mouse, rat, pig, monkey and human cell lines have been constructed. Here, we review the developmental history of CRISPR and its application in studies of human diseases. In addition, we also briefly discussed the therapeutic application of CRISPR in the near future.

Effective and precise adenine base editing in mouse zygotes

Many human genetic diseases are caused by pathogenic single nucleotide mutations. Animal models are often used to study these diseases where the pathogenic point mutations are created and/or corrected through gene editing (e.g., the CRISPR/Cas9 system) (Komoret al., 2017; Lianget al., 2017). CRISPR/Cas9-mediated gene editing depends on DNA double-strand breaks (DSBs), which can be of low efficiency and lead to indels and off-target cleavage (Kim et al., 2016). We and others have shown that base editors (BEs)may represent an attractive alternative for disease mouse model generation (Liang et al., 2017; Kim et al., 2017). Compared to CRISPR/ Cas9, cytidine base editors (CBEs) can generate C•G to T•A mutations in mouse zygotes without activating DSB repair pathways (Liang et al., 2017; Kim et al., 2017; Komor et al., 2016). In addition, CBEs showed much lower off-targets than CRISPR/Cas9 (Kim et al., 2017), making the editing process potentially safer and more controllable. Recently, adenine base editors (ABEs) that were developed from the tRNAspecific adenosine deaminase (TADA) of Escherichia coli were also reported (Gaudelli et al., 2017). As a RNA-guided programmable adenine deaminase, ABE can catalyze the conversion of A to I. Following DNA replication, base I is replaced by G, resulting in A•T to G•C conversion (Gaudelli et al., 2017; Hu et al., 2018). The development of ABEs has clearly expanded the editing capacity and application of BEs. Here, we tested whether ABEs could effectively generate disease mouse models, and found high efficiency by ABEs in producing edited mouse zygotes and mice with single-nucleotide substitutions. Unlike CBEs that can generate premature stop codons with C-T conversion (TAG, TAA or TGA), ABEs cannot produce a new stop codon to disrupt gene function via A-G conversion. We therefore targeted mRNA splice sites in order to induce gene dysfunction. Since mammalian mRNA splicing requires a 5′ GU donor and a 3′ AG acceptor at intron-exon junctions, ABEs can block mRNA splicing and hence inactivate gene function by converting splice donors and acceptors to GC and GG. We named this strategy ABEinduced mRNA splicing defect (AI-MAST). We first used ABE7.10 to target the mouse Tyr gene, whose dysfunction results in albinism in mice (Zhang et al., 2016). A gRNA was designed to target the splice donor at exon 3 of the Tyr gene, which is also predicted to be an ideal site for ABE. We then injected both ABE7.10 mRNA and the gRNA into mouse zygotes (Fig. S1A). Of the 20 embryos harvested 48 h later, 9 were edited (45.0%) with efficiencies ranging from 11.2% to 24.6% (Fig. S1B–D). In addition, 106 injected zygotes were transplanted into pseudopregnant mothers. Among the 23 pups obtained, 13 (56.5%) showed A-to-G editing with conversion frequencies of 14.6%–48.1% (Figs. S1B and S2), attesting to the feasibility of AI-MAST in generating point mutations in mice. It should be noted that we did not obtain any white-coated F0 mice, likely due to insufficient A-to-G conversion rate at the splice donor site. However, when the T1–12 F0 mouse was mated with homozygous Tyr mutant (c.655G>T, p. E219X) C57BL/6J mice (Liang et al., 2017), 2/5 (40.0%) pups were albino (Fig. S3A). Sanger sequencing results indicated that the 2 albino pups were compound heterozygous for both the ABE target site and Tyr site (c.655G>T, p. E219X) (Fig. S3B), lending support to Tyr gene dysfunction as a result of A•T to G•C conversion at the splice donor of exon 3. Furthermore, analysis of RNAs extracted from the skin of these compound heterozygous mice found significant reduction of correctly spliced Tyr mRNAs compared with Tyr mice (Fig. S3C and S3D). Both Tyr and Tyr mice showed obvious reduction of Tyr mRNA, indicating that Tyr mutant RNA is subjected to degradation by nonsense-mediated mRNA decay (NMD). These data demonstrate that AI-MAST is capable of inducing mRNA splicing defects. However, whether phenotypes associated with mRNA splicing defects can be observed in F0 mice remains unknown. To further explore one-step generation of disease mouse models using ABEs, we designed two gRNAs that targeted the splice sites at exons 61 and 66 of Dmd (Fig. 1A). These two sites were chosen because Dunchenne muscular dystrophy (DMD) remains a progressive neuromuscular degenerative disorder with no effective treatment. The largest in the human genome with 79 exons and 2.4 Mb long, the human DMD gene has recorded thousands of mutations (2,898 in the UMD-DMD database for DMD patients), including insertions, deletions, duplications and point mutations. At least 158 splice site mutations have been identified

Glycerol kinase-like proteins cooperate with Pld6 in regulating sperm mitochondrial sheath formation and male fertility

Spermatids undergo the final steps of maturation during spermiogenesis, a process that necessitates extensive rearrangement of organelles such as the mitochondria. Male infertility has been linked to mitochondrial disorder, for example, hypospermatogenesis and asthenozoospermia. However, the mechanisms that regulate mitochondrial dynamics during spermiogenesis remain largely unknown. We found the glycerol kinase (Gyk)-like proteins glycerol kinase-like 1 (Gykl1) and glycerol kinase 2 (Gk2) were specifically localized to the mitochondria in spermatids. Male mice deficient in either Gykl1 or Gk2 were infertile due to dysfunctional spermatozoa, which exhibited unregulated ATP production, disordered mitochondrial sheath formation, abnormal mitochondrial morphology, and defective sperm tail. We demonstrated that the unique C-terminal sequences found in Gykl1 and Gk2 mediated their targeting to the mitochondrial outer membrane. Furthermore, both Gykl1 and Gk2 could interact with Pld6 (MitoPLD) and induce Pld6 and phosphatidic acid (PA)-dependent mitochondrial clustering in cells. Taken together, our study has revealed previously unsuspected functions of Gyk-like proteins in spermiogenesis, providing new insight into the potential mechanisms that lead to spermatozoa dysfunction and male infertility.