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Knock-in of exogenous sequences based on CRISPR/Cas9 targeting autosomal genes and sex chromosomes in the diamondback moth, Plutella xylostella
Shanyu Li, Guifang Lin, Haoqi Wen, Haiyan Lu, Anyuan Yin, Chanqin Zheng, Feifei Li, Qingxuan Qiao, Lu Jiao, Ling Lin, Yi Yan, Xiujuan Xiang, Huang Liao, Huiting Feng, Yussuf Mohamed Salum, Minsheng You, Wei Chen, Weiyi He
2024, 23 (9): 3089-3103.   DOI: 10.1016/j.jia.2024.04.029
Abstract98)      PDF in ScienceDirect      
Genetic pest control strategies based on precise sex separation and only releasing sterile males can be accomplished by site-specific genome editing.  In the current study, we showed that the mutation of single-allele Pxfl(2)d can significantly impair the normal mating behavior and testis development in male adults of the notorious cruciferous insect pest Plutella xylostella, in addition to its known functions in the ovarian development in female adults and egg hatching.  Subsequent CRISPR/Cas9-based knock-in experiments revealed that site-specific integration of an exogenous green fluorescent protein (GFP) gene into autosomal Pxfl(2)d for labelling mutants could be achieved.  However, this gene is not a suitable target for GFP insertion to establish a genetically stable knock-in strain because of the severe decline in reproductive capacity.  We further screened for the W-chromosome-linked and Z-chromosome-linked regions to test the knock-in efficiency mediated by CRISPR/Cas9.  The results verified that both types of chromosomes can be targeted for the site-specific insertion of exogenous sequences.  We ultimately obtained a homozygous knock-in strain with the integration of both Cas9 and cyan fluorescent protein (CFP) expression cassettes on a Z-linked region in Pxylostella, which can also be used for early sex detection.  By injecting the sgRNA targeting Pxfl(2)d alone into the eggs laid by female adults of the Z-Cas9-CFP strain, the gene editing efficiency reached 29.73%, confirming the success of expressing a functional Cas9 gene.  Taken together, we demonstrated the feasibility of the knock-in of an exogenous gene to different genomic regions in Pxylostella, while the establishment of a heritable strain required the positioning of appropriate sites.  This study provides an important working basis and technical support for further developing genetic strategies for insect pest control.
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Biology of Hippo signaling pathway: Skeletal muscle development and beyond

Shuqi Qin, Chaocheng Li, Haiyan Lu, Yulong Feng, Tao Guo, Yusong Han, Yongsheng Zhang, Zhonglin Tang
2024, 23 (6): 1825-1838.   DOI: 10.1016/j.jia.2023.09.031
Abstract123)      PDF in ScienceDirect      

Global demand for farm animals and their meat products i.e., pork, chicken and other livestock meat, is steadily incresing. With the ongoing life science research and the rapid development of biotechnology, it is a great opportunity to develop advanced molecular breeding markers to efficiently improve animal meat production traits.  Hippo is an important study subject because of its crucial role in the regulation of organ size.  In recent years, with the increase of research on Hippo signaling pathway, the integrative application of multi-omics technologies such as genomics, transcriptomics, proteomics, and metabolomics can help promote the in-depth involvement of Hippo signaling pathway in skeletal muscle development research.  The Hippo signaling pathway plays a key role in many biological events, including cell division, cell migration, cell proliferation, cell differentiation, cell apoptosis, as well as cell adhesion, cell polarity, homeostasis, maintenance of the face of mechanical overload, etc.  Its influence on the development of skeletal muscle has important research value for enhancing the efficiency of animal husbandry production.  In this study, we traced the origin of the Hippo pathway, comprehensively sorted out all the functional factors found in the pathway, deeply analyzed the molecular mechanism of its function, and classified it from a novel perspective based on its main functional domain and mode of action.  Our aim is to systematically explore its regulatory role throughout skeletal muscle development.  We specifically focus on the Hippo signaling pathway in embryonic stem cell development, muscle satellite cell fate determination, myogenesis, skeletal muscle meat production and organ size regulation, muscle hypertrophy and atrophy, muscle fiber formation and its transformation between different types, and cardiomyocytes.  The roles in proliferation and regeneration are methodically summarized and analyzed comprehensively.  The summary and prospect of the Hippo signaling pathway within this article will provide ideas for further improving meat production and muscle deposition and developing new molecular breeding technologies for livestock and poultry, which will be helpful for the development of animal molecular breeding.

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