Yuxi Chen

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.

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.

Role of a Novel Functional Variant in the PPP2R1A Promoter on the Regulation of PP2A-Aalpha and the Risk of Hepatocellular Carcinoma

Previously, we identified the genetic variant −241 (−/G) (rs11453459) in the PP2A-Aα gene (PPP2R1A) promoter and demonstrated that this variant influences the DNA-binding affinity of nuclear factor-kappa B (NF-κB). In this study, we further confirmed that the transcriptional activity of PPP2R1A may be regulated by NF-κB through the functional genetic variant −241 (−/G). Moreover, we also demonstrated that the methylation status of CpG islands in the promoter of PPP2R1A influences the activity of this gene promoter. Few studies have examined the role of this −241 (−/G) variant in genetic or epigenetic regulation in hepatocellular carcinoma (HCC). To investigate whether this functional variant in the PPP2R1A promoter is associated with the risk of HCC and confirm the function of the −241 (−/G) variant in the HCC population, we conducted a case-control study involving 251 HCC cases and 252 cancer-free controls from a Han population in southern China. Compared with the −241 (−−) homozygote, the heterozygous −241 (−G) genotype (adjusted OR  = 0.32, 95% confidence interval (CI)  = 0.17–0.58, P<0.001) and the −241 (−G)/(GG) genotypes (adjusted OR  = 0.38, 95% CI  = 0.22–0.67, P  = 0.001) were both significantly associated with a reduced risk of HCC. Stratification analysis indicated that the protective role of −241 (−G) was more pronounced in individuals who were ≤ 40 years of age, female and HBV-negative. Our data suggest that the transcriptional activity of PPP2R1A is regulated by NF-κB through the −241 (−/G) variant and by the methylation of the promoter region. Moreover, the functional −241 (−/G) variant in the PPP2R1A promoter contributes to the decreased risk of HCC. These findings contribute novel information regarding the gene transcription of PPP2R1A regulated by the polymorphism and methylation in the promoter region through genetic and epigenetic mechanisms in hepatocarcinogenesis.

Cold-inducible RNA-binding protein CIRP/hnRNP A18 regulates telomerase activity in a temperature-dependent manner

The telomerase is responsible for adding telomeric repeats to chromosomal ends and consists of the reverse transcriptase TERT and the RNA subunit TERC. The expression and activity of the telomerase are tightly regulated, and aberrant activation of the telomerase has been observed in >85% of human cancers. To better understand telomerase regulation, we performed immunoprecipitations coupled with mass spectrometry (IP-MS) and identified cold inducible RNA-binding protein (CIRP or hnRNP A18) as a telomerase-interacting factor. We have found that CIRP is necessary to maintain telomerase activities at both 32°C and 37°C. Furthermore, inhibition of CIRP by CRISPR-Cas9 or siRNA knockdown led to reduced telomerase activities and shortened telomere length, suggesting an important role of CIRP in telomere maintenance. We also provide evidence here that CIRP associates with the active telomerase complex through direct binding of TERC and regulates Cajal body localization of the telomerase. In addition, CIRP regulates the level of TERT mRNAs. At the lower temperature, TERT mRNA is upregulated in a CIRP-dependent manner to compensate for reduced telomerase activities. Taken together, these findings highlight the dual roles that CIRP plays in regulating TERT and TERC, and reveal a new class of telomerase modulators in response to hypothermia conditions.

Identification and Functional Analysis of Variant Haplotypes in the 5′-Flanking Region of Protein Phosphatase 2A-Bδ Gene

Serine-threonine protein phosphatase 2A (PP2A) is a trimeric holoenzyme that plays an integral role in the regulation of cell growth, differentiation, and apoptosis. The substrate specificity and (sub)cellular localization of the PP2A holoenzymes are highly regulated by interaction with a family of regulatory B subunits (PP2A-Bs). The regulatory subunit PP2A-B/PR55δ (PP2A-Bδ) is involving in the dephosphorylation of PP2A substrates and is crucial for controlling entry into and exit from mitosis. The molecular mechanisms involved in the regulation of expression of PP2A-Bδ gene (PPP2R2D) remain largely unknown. To explore genetic variations in the 5′-flanking region of PPP2R2D gene as well as their frequent haplotypes in the Han Chinese population and determine whether such variations have an impact on transcriptional activity, DNA samples were collected from 70 healthy Chinese donors and sequenced for identifying genetic variants in the 5′-flanking region of PPP2R2D. Four genetic variants were identified in the 1836 bp 5′-flanking region of PPP2R2D. Linkage disequilibrium (LD) patterns and haplotype profiles were constructed for the genetic variants. Using serially truncated human PPP2R2D promoter luciferase constructs, we found that a 601 bp (−540 nt to +61 nt) fragment constitutes the core promoter region. The subcloning of individual 5′-flanking fragment revealed the existence of three haplotypes in the distal promoter of PPP2R2D. The luciferase reporter assay showed that different haplotypes exhibited distinct promoter activities. The EMSA revealed that the −462 G>A variant influences DNA-protein interactions involving the nuclear factor 1 (NF1). In vitro reporter gene assay indicated that cotransfection of NF1/B expression plasmid could positively regulate the activity of PPP2R2D proximal promoter. Introduction of exogenous NF1/B expression plasmid further confirmed that the NF1 involves in the regulation of PPP2R2D gene expression. Our findings suggest that functional genetic variants and their haplotypes in the 5′-flanking region of PPP2R2D are critical for transcriptional regulation of PP2A-Bδ.

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.

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.

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.

Testis-specific Lypd9 is dispensable for spermatogenesis in mouse

1Key Laboratory of Reproductive Medicine of Guangdong Province, the First Affiliated Hospital and School of Life Sciences and the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China 2Key Laboratory of Gene Engineering of the Ministry of Education and State Key Laboratory of Biocontrol, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, China 3Key Laboratory of Reproductive Medicine of Guangdong Province, Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China