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Generation of CRISPR/Cas9-induced BMD mice

DM Daigo Miyazaki
MS Mitsuto Sato
NS Naoko Shiba
TY Takahiro Yoshizawa
AN Akinori Nakamura

Last updated date: Jun 18, 2025 Views: 330 Forks: 0

original An abbreviated version of this protocol was published in eLife in Mar, 2025
Becker muscular dystrophy mice showed site-specific decay of type IIa fibers with capillary change in skeletal muscle
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Generation of CRISPR/Cas9-induced BMD mice

To introduce the mutations d45–48, d45–47, and d45–49 in the mouse Dmd, we designed four gRNA 

corresponding to intron sequences 44, 47, 48, and 49 (gRNA- 44, -47, -48, and -49, respectively). 

Next, we electroporated two gRNA at the combination of gRNA- 44 and -47 to generate d45–47, 

gRNA- 44 and -48 to generate d45–48, and gRNA- 44 and -49 to generate d45–49, with Cas9- nuclease, into embryos from C57BL/6J female mice. (These descriptions are excerpts from eLife (DOI: doi.org/10.7554/eLife.100665.3))

 

gRNA sequences (5' to 3')

gRNA- 44: (DMD_44-45_crRNA_1)

/AlTR1/rGrUrUrUrCrArCrUrGrUrGrUrCrGrGrUrUrUrArArGrUrUrUrUrArGrArGrCrUrArUrGrCrU/AlTR2/

gRNA- 47: (DMD_47-48_crRNA_1)

/AlTR1/rUrCrCrArUrUrGrArCrArCrUrArGrUrCrGrGrCrArGrUrUrUrUrArGrArGrCrUrArUrGrCrU/AlTR2/

gRNA- 48: (DMD_48-49_crRNA_2)

/AlTR1/rArGrArArCrArUrUrUrCrArCrCrCrUrArCrGrArUrGrUrUrUrUrArGrArGrCrUrArUrGrCrU/AlTR2/

gRNA- 49: (DMD_49-50_crRNA_1)

/AlTR1/rArArUrUrArCrArGrUrCrGrArUrCrArCrArUrCrCrGrUrUrUrUrArGrArGrCrUrArUrGrCrU/AlTR2/

 

To generation d45-47 BMD mice: gRNA- 44 + gRNA- 47 + tracrRNA + Cas9- Nuclease

Generation of d45-48 BMD mice: gRNA- 44 + gRNA- 48 + tracrRNA + Cas9- Nuclease

Generation of d45-49 BMD mice: gRNA- 44 + gRNA- 49 + tracrRNA + Cas9- Nuclease

Copyright: © 2025 The Author(s); This is an open-access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Miyazaki, D, Sato, M, Shiba, N, Yoshizawa, T and Nakamura, A(2025). Generation of CRISPR/Cas9-induced BMD mice. Bio-protocol Preprint. bio-protocol.org/prep2829.
  2. Miyazaki, D., Sato, M., Shiba, N., Yoshizawa, T. and Nakamura, A.(2025). Becker muscular dystrophy mice showed site-specific decay of type IIa fibers with capillary change in skeletal muscle. eLife. DOI: 10.7554/eLife.100665

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