Zebrafish Embryonic Slow Muscle Is a Rapid System for Genetic Analysis of Sarcomere Organization by CRISPR/Cas9, but Not NgAgo
Zebrafish embryonic slow muscle cells, with their superficial localization and clear sarcomere organization, provide a useful model system for genetic analysis of muscle cell differentiation and sarcomere assembly. To develop a quick assay for testing CRISPR-mediated gene editing in slow muscles of zebrafish embryos, we targeted a red fluorescence protein (RFP) reporter gene specifically expressed in slow muscles of myomesin-3-RFP (Myom3-RFP) zebrafish embryos. We demonstrated that microinjection of RFP-sgRNA with Cas9 protein or Cas9 mRNA resulted in a mosaic pattern in loss of RFP expression in slow muscle fibers of the injected zebrafish embryos. To uncover gene functions in sarcomere organization, we targeted two endogenous genes, slow myosin heavy chain-1 (smyhc1) and heat shock protein 90 α1 (hsp90α1), which are specifically expressed in zebrafish muscle cells. We demonstrated that injection of Cas9 protein or mRNA with respective sgRNAs targeted to smyhc1 or hsp90a1 resulted in a mosaic pattern of myosin thick filament disruption in slow myofibers of the injected zebrafish embryos. Moreover, Myom3-RFP expression and M-line localization were also abolished in these defective myofibers. Given that zebrafish embryonic slow muscles are a rapid in vivo system for testing genome editing and uncovering gene functions in muscle cell differentiation, we investigated whether microinjection of Natronobacterium gregoryi Argonaute (NgAgo) system could induce genetic mutations and muscle defects in zebrafish embryos. Single-strand guide DNAs targeted to RFP, Smyhc1, or Hsp90α1 were injected with NgAgo mRNA into Myom3-RFP zebrafish embryos. Myom3-RFP expression was analyzed in the injected embryos. The results showed that, in contrast to the CRISPR/Cas9 system, injection of the NgAgo-gDNA system did not affect Myom3-RFP expression and sarcomere organization in myofibers of the injected embryos. Sequence analysis failed to detect genetic mutations at the target genes. Together, our studies demonstrate that zebrafish embryonic slow muscle is a rapid model for testing gene editing technologies in vivo and uncovering gene functions in muscle cell differentiation.
KeywordsSlow myofibers Myomesin-3-RFP Hsp90α1 CRISPR/Cas9 NgAgo
This research was supported by a seed fund from the University of Maryland Baltimore and Chinese National Science Foundation (31230076). MC and YFS were supported by China Scholarship Council.
Conceived and designed the experiments: MC and SD. Performed the experiments: MC, YFS, and SD. Analyzed the data: MC, JZ, ZT, and SD. Wrote the paper: MC, JZ, and SD.
Compliance with Ethical Standards
All animal studies were carried out according to the guideline for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the University of Maryland (Permit Number 0516005).
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