Genetics and Genomics

Production of non-mosaic genome edited porcine embryos by injection of CRISPR/Cas9 into germinal vesicle oocytes Publication date: Available online 20 July 2019 Source: Journal of Genetics and Genomics Author(s): Xiaohu Su, Wei Chen, Qingqing Cai, Puping Liang, Yaosheng Chen, Peiqing Cong, Junjiu Huang Abstract: Genetically modified pigs represent a great promise for generating models of human diseases and producing new breeds. Generation of genetically edited pigs using somatic cell nuclear transfer (SCNT) or zygote cytoplasmic microinjection is a tedious process due to the low developmental rate or mosaicism of the founder (F0). Herein, we developed a method termed germinal vesicle oocyte gene editing (GVGE) to produce non-mosaic porcine embryos by editing maternal alleles during the GV to MⅡ transition. Injection of Cas9 mRNA and X-linked Dmd gene-specific gRNA into GV oocytes did not affect their developmental potential. The MⅡ oocytes edited during in vitro maturation (IVM) could develop into blastocysts after parthenogenetic activation (PA) or in vitro fertilization (IVF). Genotyping results indicated that the maternal gene X-linked Dmd could be efficiently edited during oocyte maturation. Up to 81.3% of the edited IVF embryos were non-mosaic Dmd gene mutant embryos. In conclusion, GVGE might be a valuable method for the generation of non-mosaic maternal allele edited F0 embryos in a short simple step.

Gene redundancy and gene compensation: an updated view Publication date: Available online 19 July 2019 Source: Journal of Genetics and Genomics Author(s): Jinrong Peng Abstract Gene knockdown approaches using antisense oligo nucleotides or analogs such as siRNAs and morpholinos have been widely adopted to study gene functions although the off-target issue has been always a concern in these studies. On the other hand, classic genetic analysis relies on the availability of loss-of-function or gain-of-function mutants. The fast development of gene editing technologies such as TALEN and CRISPR/Cas9 has greatly facilitated the generation of null mutants for the functional studies of target genes in a variety of organism including zebrafish. Surprisingly, an unexpected discrepancy was observed between morphant phenotype and mutant phenotype for many genes in zebrafish, i.e., while the morphant often displays an obvious phenotype, the corresponding null mutant appears relatively normal or only exhibits a mild phenotype due to gene compensation. Two recent reports have partially answered this intriguing question by showing that a pre-mature termination and homologous sequence are required to elicit the gene compensation and the histone modifying complex COMPASS is involved in activating the expression of the compensatory genes. Here, I summarize these exciting new progress and try to redefine the concept of genetic compensation and gene compensation.

Rare variants in novel and known genes associated with primary angle closure glaucoma based on whole exome sequencing of 549 probands Publication date: Available online 10 July 2019 Source: Journal of Genetics and Genomics Author(s): Wenmin Sun, Xueshan Xiao, Shiqiang Li, Jiamin Ouyang, Xueqing Li, Xiaoyun Jia, Xing Liu, Qingjiong Zhang

OsGCD1, a novel player in rice intine construction Publication date: Available online 25 June 2019 Source: Journal of Genetics and Genomics Author(s): Xiaorong Huang, Mengmeng Run, Meng-Xiang Sun

Dissecting PCNA function with a systematically designed mutant library in yeast Publication date: Available online 24 June 2019 Source: Journal of Genetics and Genomics Author(s): Qingwen Jiang, Weimin Zhang, Chenghao Liu, Yicong Lin, Qingyu Wu, Junbiao Dai Abstract Proliferating cell nuclear antigen (PCNA), encoded by POL30 in Saccharomyces cerevisiae, is a key component of DNA metabolism. Here, a library consisting of 304 PCNA mutants was designed and constructed to probe the contribution of each residue to the biological function of PCNA. Five regions with elevated sensitivity to DNA damaging reagents were identified using high-throughput phenotype screening. Using a series of genetic and biochemical analyses, we demonstrated that one particular mutant, K168A, has defects in the DNA damage tolerance (DDT) pathway by disrupting the interaction between PCNA and Rad5. Subsequent domain analysis showed that the PCNA-Rad5 interaction is a prerequisite for the function of Rad5 in DDT. Our study not only provides a resource in the form of a library of versatile mutants to study the functions of PCNA, but also reveals a key residue on PCNA (K168) which highlights the importance of the PCNA-Rad5 interaction in the template switching (TS) pathway.

A modified high-efficiency thermal asymmetric interlaced PCR method for amplifying long unknown flanking sequences Publication date: Available online 23 June 2019 Source: Journal of Genetics and Genomics Author(s): Jiantao Tan, Qi Gong, Suize Yu, Yuke Hou, Dongchang Zeng, Qinlong Zhu, Yao-Guang Liu

Nlrc3-like is required for microglia maintenance in zebrafish Publication date: Available online 22 June 2019 Source: Journal of Genetics and Genomics Author(s): Tienan Wang, Bo Yan, Liang Lou, Xi Lin, Tao Yu, Shuting Wu, Qing Lu, Wei Liu, Zhibin Huang, Mingjie Zhang, Wenqing Zhang, Zilong Wen Abstract Microglia are tissue-resident macrophages residing in the central nervous system (CNS) and play critical roles in removing cellular debris and infectious agents as well as regulating neurogenesis and neuronal activities. Yet, the molecular basis underlying the establishment of microglia pool and the maintenance of their homeostasis in the CNS remain largely undefined. Here we report the identification and characterization of a mutant zebrafish, which harbors a point mutation in the nucleotide-binding oligomerization domain (NOD) like receptor gene nlrc3-like, resulting in the loss of microglia in a temperature sensitive manner. Temperature shift assay reveals that the late onset of nlrc3-likedeficiency leads to excessive microglia cell death. Further analysis shows that the excessive microglia death in nlrc3-like deficient mutants is attributed, at least in part, to aberrant activation of canonical inflammasome pathway. Our study indicates that proper regulation of inflammasome cascade is critical for the maintenance of microglia homeostasis.

Mutations in TP53, ZNF750, and RB1 typify ocular sebaceous carcinoma Publication date: Available online 21 June 2019 Source: Journal of Genetics and Genomics Author(s): Yongyang Bao, J. Eva Selfridge, Janet Wang, Yiqing Zhao, Junqi Cui, Kishore Guda, Zhenghe Wang, Yanbo Zhu

Pyramiding of the dep1-1 and NAL1NJ6 alleles achieves sustainable improvements in nitrogen-use efficiency and grain yield in japonica rice breeding Publication date: Available online 1 June 2019 Source: Journal of Genetics and Genomics Author(s): Xiaopeng Xu, Kun Wu, Ruineng Xu, Jianping Yu, Jing Wang, Ying Zhao, Yun Wang, Wenzhen Song, Shuoxun Wang, Zhi Gao, Yongjia Zhong, Xinxin Li, Hong Liao, Xiangdong Fu

RSBP15 interacts with and stabilizes dRSPH3 during sperm axoneme assembly in Drosophila Publication date: Available online 1 June 2019 Source: Journal of Genetics and Genomics Author(s): Ya Wang, Rui Xu, Yiwei Cheng, Haowei Cao, Zibin Wang, Tianyu Zhu, Jiayin Jiang, Hao Zhang, Chang Wang, Lin Qi, Mingxi Liu, Xuejiang Guo, Juan Huang, Jiahao Sha Abstract Flagellum in sperm is composed of over 200 different proteins and is essential for sperm motility. In particular, defects in the assembly of the radial spoke in the flagellum result in male infertility due to loss of sperm motility. However, mechanisms regulating radial spoke assembly remain unclear in metazoans. Here, we identified a novel Drosophila protein radial spoke binding protein 15 (RSBP15) which plays an important role in regulating radial spoke assembly. Loss of RSBP15 results in complete lack of mature sperms in seminal vesicles (SVs), asynchronous individualization complex (IC) and defective “9 + 2” structure in flagella. RSBP15 is colocalized with dRSPH3 in sperm flagella, and interacts with dRSPH3 through its DD_R_PKA superfamily domain which is important for the stabilization of dRSPH3. Moreover, loss of dRSPH3, as well as dRSPH1, dRSPH4a and dRSPH9, showed similar phenotypes to rsbp15KOmutant. Together, our results suggest that RSBP15 acts in stabilizing the radial spoke protein complex to anchor and strengthen the radial spoke structures in sperm flagella.