Yuning Song

CRISPR/Cas9-mediated GJA8 knockout in rabbits recapitulates human congenital cataracts.

Cataracts are the leading cause of vision loss in the world, although surgical treatment can restore vision in cataract patients. Until now, there have been no adequate animal models for in vivo studies of artificial lens safety and drug interactions. Genetic studies have demonstrated that GJA8 is involved in maintaining lens opacity and proper lens development. In this study, a cataract model with GJA8 gene knockout was developed via co-injection of Cas9/sgRNA mRNA into rabbit zygotes. Our results showed that gene mutation efficiency in the GJA8 locus reached 98.7% in embryos and 100% in pups, demonstrating that the Cas9/sgRNA system is a highly efficient tool for gene editing in rabbits. In agreement with other studies, our genetic and histology results showed that impaired GJA8 function caused microphthalmia, small lens size and cataracts. In summary, our novel rabbit model of cataracts will be an important drug-screening tool for cataract prevention and treatment.

Aberrant expression of Igf2/H19 in porcine parthenogenetic fetuses and placentas

The aberrant expression of imprinted genes induces parthenogenetic fetal and placental dysplasia, thus leading to failures in embryonic development. Igf2 and H19 are co-expressed in endoderm and mesoderm-derived tissues and play an important role in normal embryo and extraembryonic development. In this study, the expression and methylation of Igf2/H19 in porcine parthenogenetic fetuses and placentas which had grown 28 days was examined first time to further characterize mammalian parthenogenesis. Weight and morphological comparisons were conducted between parthenogenetic embryos on Day 28 and normal fertilized embryos (control). The results indicated that parthenogenetic fetuses and placentas had smaller weights and volumes than those of the control. In addition, quantitative RT-PCR (qRT-PCR) analysis was performed to determine Igf2/H19 expression levels, showing that the expression of H19 was up-regulated, while Igf2 expression was almost undetectable in both parthenogenetic fetuses and placentas. As a potential mechanism underlying this disrupted expression, the methylation of Igf2/H19 DMR3 was detected using bisulfite sequencing PCR analysis, which revealed the significant hypomethylation of DMR3 in parthenogenetic fetuses and placentas. These results suggest that disruption of Igf2/H19 expression in parthenogenetic fetuses and placentas contributes to implantation failure and/or abortion in swine parthenogenesis, which might be associated with differential methylation patterns in the imprinting control region of imprinted genes.

Disruption of imprinted gene expression and DNA methylation status in porcine parthenogenetic fetuses and placentas.

Parthenogenetically activated oocytes cannot develop to term in mammals due to the lack of paternal gene expression and failed X chromosome inactivation (XCI). To further characterize porcine parthenogenesis, the expression of 18 imprinted genes was compared between parthenogenetic (PA) and normally fertilized embryos (Con) using quantitative real-time PCR (qRT-PCR). The results revealed that maternally expressed genes were over-expressed, whereas paternally expressed genes were significantly reduced in PA fetuses and placentas. The results of bisulfite sequencing PCR (BSP) demonstrated that PRE-1 and Satellite were hypermethylated in both Con and PA fetuses and placentas, while XIST DMRs were hypomethylated only in PA samples. Taken together, these results suggest that the aberrant methylation profile of XIST DMRs and abnormal imprinted gene expression may be responsible for developmental failure and impaired growth in porcine parthenogenesis.

Conservation of imprinting of Neuronatin ( Nnat ) in rabbits

Although the expression and epigenetic status of imprinted genes have been extensively studied in a number of species, less is known about the genomic imprinting in rabbits. Neuronatin (Nnat) plays significant roles in the brain development and metabolic regulation and has been identified to be imprinted and paternally expressed in humans, mice and pigs; however, it has not yet been investigated in rabbits. In this study, we confirmed the expression of two isoforms of the rabbit Nnat (Nnat-a and Nnat-β) identified in Genbank and Ensembl by quantitative real-time PCR. In addition, we also determined the methylation profile of the CpG island in the promoter region of the rabbit Nnat using bisulfite sequencing PCR and combined bisulfite restriction analysis. Here, we provide the first evidence that Nnat has two transcripts in rabbit. Additionally, the CpG island located in the promoter region shows oocyte-specific methylation and may be the differentially methylated region of Nnat in rabbits.

CRISPR/Cas9-mediated mutation of tyrosinase (Tyr) 3′ UTR induce graying in rabbit

The 3′ untranslated regions (UTRs), located at the end of mRNA molecules, are believed to play a role in RNA replication and/or protein translation. Mutations in the tyrosinase (Tyr) gene are known to cause recessive albinism in humans and other species. In this study, to test whether the CRISPR/Cas9 system works on the mutation of the UTRs regulatory region in rabbit, the 3′ UTR of the rabbit Tyr gene was deleted by a dual sgRNA directed CRISPR/Cas9 system. As expected, gray coat color and reduced melanin in hair follicles and irises was found in the mutated rabbit, thus increasing confidence in the association of the mutation of the Tyr 3′ UTR with graying in rabbit. The graying phenotype was also found in the F1 generation, suggesting that the mutated allele can be stably inherited by the offspring. Thus, we provide the first evidence that reduced melanin and graying can be caused by deletion of the Tyr 3′ UTR in rabbits. Additionally, CRISPR/Cas9-mediated large fragment deletions can facilitate genotype to phenotype studies of UTRs or non-coding RNAs in future.

D-repeat in the XIST gene is required for X chromosome inactivation

ABSTRACT XIST is a long non-coding RNA, which expressed exclusively from the inactive X chromosome. Although it has been revealed that the A-repeat contributes to the X chromosome inactivation (X-inactivation), the role of the longest D-repeat has not yet been investigated. Here, a sgRNA directed CRISPR/Cas9 system which have multiple target sites within repeat D of XIST, were used to generate D-repeat deletion and studied its roles on X-inactivation. The results showed that the deletion of D-repeat caused a significantly decreased expression of XIST, and up regulated expression of X-linked genes, suggesting that the D-repeat may play an important role in the regulation of XIST expression and silencing of the X-linked genes, which could provide a new idea in the molecular mechanisms of X-inactivation.

Highly efficient RNA-guided base editing in rabbit

Cytidine base editors (CBEs) and adenine base editors (ABEs), composed of a cytidine deaminase or an evolved adenine deaminase fused to Cas9 nickase, enable the conversion of C·G to T·A or A·T to G·C base pair in organisms, respectively. Here, we show that BE3 and ABE7.10 systems can achieve a targeted mutation efficiency of 53–88% and 44–100%, respectively, in both blastocysts and Founder (F0) rabbits. Meanwhile, this strategy can be used to precisely mimic human pathologies by efficiently inducing nonsense or missense mutations as well as RNA mis-splicing in rabbit. In addition, the reduced frequencies of indels with higher product purity are also determined in rabbit blastocysts by BE4-Gam, which is an updated version of the BE3 system. Collectively, this work provides a simple and efficient method for targeted point mutations and generation of disease models in rabbit.Base editors can make targeted changes without inducing a double-stranded break. Here, the authors apply the BE3 and ABE7.10 systems to rabbit to create highly efficient targeted base substitutions and various mutation types, and show reduced frequency of undesired by-products with the updated BE4-Gam system.

CRISPR/Cas9–Mediated Mutation of αA-Crystallin Gene Induces Congenital Cataracts in Rabbits

Purpose The present study aimed to investigate the role of the αA-crystallin gene in inducing congenital cataracts in rabbits and to construct a novel animal model for characterization and pathologic analysis of congenital cataracts for future research. Methods We generated αA-crystallin gene knockout rabbits with congenital cataracts by coinjection of Cas9 mRNA and sgRNA into zygotes. Cataract phenotypes were investigated in a repeated study of 19 F0-generation and 11 F1-generation rabbits with αA-crystallin gene mutations. Heritability was analyzed by PCR, sequencing, slim lamp, hematoxylin eosin staining, immunohistochemistry, and Western blot. Results We found αA-crystallin gene mutations in all 19 F0-generation pups (100%) with indel mutations in the αA-crystallin gene ranging from 3 to 52 bp. Off-target assay revealed that none of the potential off-target sites exhibited mutations, demonstrating that off-target mutagenesis was not induced by cytoplasmic microinjection of in vitro-transcribed Cas9 mRNA. Slim lamp assay revealed that 15 of 19 live pups (78.9%) exhibited typical phenotypes, including congenital cataracts, microphthalmia, obscurity, and early atrophy of the lens, and failed differentiation of lens fibers. Histologic hematoxylin and eosin staining showed that αA-crystallin gene knockout rabbits exhibited smaller lenses. Production of the αA-crystallin protein was determined to be dramatically reduced in αA-crystallin gene knockout rabbits. We induced αA-crystallin gene mutations and phenotypes in F1-generation rabbits. Conclusions Our data suggest that CRISPR/Cas9-mediated mutation of the αA-crystallin gene in rabbits recapitulates phenotypes of congenital cataracts, microphthalmia, obscurity, and early atrophy of the lens, and failed differentiation of lens fibers. These findings suggest the possibility of a new animal model of congenital cataracts, which should be used to further investigate the association between mutations in αA-crystallin gene and congenital cataracts in humans.

Germ cell‐specific expression of Cre recombinase using the VASA promoter in the pig

The Cre–loxP system is a powerful tool for genetic analysis of distinct cell lineages and tissue‐specific gene knockout in animal models. VASA is specifically expressed in reproductive tissues, and is known to play important roles in spermatogenesis and germ‐cell growth. In this study, Cre recombinase transgenic pigs under the control of the VASA promoter were generated by somatic cell nuclear transfer. Germ cell‐specific expression of Cre recombinase in VASA‐Cre transgenic pigs was shown by western blotting and immunohistochemistry. VASA‐Cre transgenic pigs will be a useful tool for germ cell‐specific gene knockout and a disease model for disorders of the reproductive system.

Mutation of the Sp1 binding site in the 5’ flanking region of SRY causes sex reversal in rabbits

Sex-determining region Y is a crucial gene that initiates male sex determination in mammals. Mutations of the Sp1-binding site in the 5′ flanking region of SRY are associated with clinical male-to-female sex reversal syndrome, although such occurrences are rare and, until now, have not been reported in animal models. In this study, we mutated Sp1-binding sites in the 5′ flanking region of the rabbit SRY gene using the CRISPR/Cas9 system. As expected, the SRY-Sp1 knockout rabbits had female external and internal genitalia and exhibited normal female copulatory behaviors, but they were infertile, and the adults displayed reduced follicles. Interestingly, we successfully obtained offspring from sex-reversed SRY-Sp1 knockout rabbits using embryo transfer. In summary, our study demonstrates that Sp1 is a major regulator in SRY gene transcription, and mutations of the Sp1 binding sites (Sp1-B and Sp1-C) in the 5′ flanking region of SRY induce sex reversal in rabbits, which can be used as targets for clinical research of male-to-female sex reversal syndrome. Additionally, we provide the first evidence that sex reversal syndrome patients have the potential to become pregnant with the use of embryo transfer.