Combined CRISPR toolkits reveal the domestication landscape and function of the ultra-long and highly repetitive silk genes. (1st March 2023)

• Record Type: Journal Article
• Authors: Lu, Wei; Ma, Sanyuan; Sun, Le; Zhang, Tong; Wang, Xiaogang; Feng, Min; Wang, Aoming; Shi, Run; Jia, Ling; Xia, Qingyou
• Is Part Of: Acta biomaterialia. Volume 158(2023)
• Page Start: 190
• Page End: 202
• Publication Date: 2023-03-01
• Subjects: Highly repetitive sequence -- FibH gene -- CRISPR/Cas9 -- SMRT sequencing -- Mechanical properties; CRISPR Clustered Regularly Interspaced Short Palindromic Repeats -- gRNA guide RNA -- CACS Cas9-assistedcloning andSMRT sequencing of long repetitive sequences -- FibH fibroin heavy chain -- FibL fibroin light chain -- NTD N-terminal domain -- CTD C-terminal domain -- gDNA genomic DNA -- ONT Oxford Nanopore Technologies; Biomedical materials -- Periodicals; 610.28
• DOI: 10.1016/j.actbio.2022.12.063
• Languages: English
• ISSNs: 1742-7061

Abstract

Abstract: Highly repetitive sequences play a major structural and function role in the genome. In the present study, we developed C as9-a ssisted c loning and S MRT sequencing of long repetitive sequences (CACS) to sequence and manipulate highly repetitive genes from eukaryotic genomes. CACS combined Cas9-mediated cleavage of a target segment from an intact genome, Gibson assembly cloning, and PacBio SMRT sequencing. Applying CACS, we directly cloned and sequenced the complete sequences of fibroin heavy chain ( FibH ) genes from 17 domesticated ( Bombyx mori ) and 7 wild ( Bombyx mandarina ) silkworms. Our analysis revealed the unique fine structure organization, genetic variations, and domestication dynamics of FibH . We also demonstrated that the length of the repetitive regions determined the mechanical properties of silk fiber, which was further confirmed by Cas9 editing of FibH . CACS is a simple, robust, and efficient approach, providing affordable accessibility to highly repetitive regions of a genome. Statement of significance: Silkworm silk is the earliest and most widely used animal fiber, and its excellent performance mainly depends on the fibroin heavy chain (FibH) protein. The FibH gene is the main breakthrough in understanding the formation mechanism and improvement of silk fiber. In the study, we developed a CACS method for characterizing the fine structure and domestication landscape of 24 silkworm FibH genes. We used CRISPR/Cas9 to edit the repetitive sequence of FibH genes, revealing the relationship between FibH genes and mechanical properties of silkworm silk. Our study is helpful in modifying silk genes to manipulate other valuable highly repetitive sequences, and provides insight for silkworm breeding. Graphical abstract: Image, graphical abstract