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CRISPR/Cas9 Mediated High Efficiency Knockout of Myosin Essential Light Chain Gene in the Pacific Oyster (Crassostrea Gigas)

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Abstract

Pacific oyster (Crassostrea gigas) is one of the most widely cultivated shellfish species in the world. Because of its economic value and complex life cycle involving drastic changes from a free-swimming larva to a sessile juvenile, C. gigas has been used as a model for developmental, environmental, and aquaculture research. However, due to the lack of genetic tools for functional analysis, gene functions associated with biological or economic traits cannot be easily determined. Here, we reported a successful application of CRISPR/Cas9 technology for knockout of myosin essential light chain gene (CgMELC) in C. gigas. C. gigas embryos injected with sgRNAs/Cas9 contained extensive indel mutations at the target sites. The mutant larvae showed defective musculature and reduced motility. In addition, knockout of CgMELC disrupted the expression and patterning of myosin heavy chain positive myofibers in larvae. Together, these data indicate that CgMELC is involved in larval muscle contraction and myogenesis in oyster larvae.

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Funding

This study was supported by grants from National Key R&D Program of China (2018YFD0900200), National Natural Science Foundation of China (31972789), Shandong Province (2017LZGC009), and the Ocean University of China-Auburn University Joint Research Center for Aquaculture and Environmental Science.

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Correspondence to Qi Li.

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10126_2020_10016_MOESM1_ESM.tif

Supplementary file1: Off-target analyze of sgRNAs. Three potential off target sites for sgRNAs were screened and identified by sanger sequence, and no evidence for off-target mutagenesis of sgRNAs (TIF 451 KB)

Supplementary file2: The motor ability of larvae injected with CgMELC-sgRNA-1/2 and Cas9 mRNA. Knockout of CgMELC resulted in significant reduction of motor ability (MP4 11618 KB)

Supplementary file3: The motor ability of wild type larvae (MP4 12934 KB)

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Li, H., Yu, H., Du, S. et al. CRISPR/Cas9 Mediated High Efficiency Knockout of Myosin Essential Light Chain Gene in the Pacific Oyster (Crassostrea Gigas). Mar Biotechnol 23, 215–224 (2021). https://doi.org/10.1007/s10126-020-10016-1

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  • DOI: https://doi.org/10.1007/s10126-020-10016-1

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