Generation of a zebrafish knock-in line expressing MYC-tagged Sox11a using CRISPR/Cas9 genome editing

Laura A. Krueger, Ann C. Morris

Research output: Contribution to journalArticlepeer-review

Abstract

Advances in CRISPR-Cas9 genome editing technology have strengthened the role of zebrafish as a model organism for genetics and developmental biology. These tools have led to a significant increase in the production of loss-of-function mutant zebrafish lines. However, the generation of precisely edited knock-in lines has remained a significant challenge in the field due to the decreased efficiency of homology directed repair (HDR). In this study, we overcame some of these challenges by combining available design tools and synthetic, commercially available CRISPR reagents to generate a knock-in line carrying an in-frame MYC epitope tag at the sox11a locus. Zebrafish Sox11a is a transcription factor with critical roles in organogenesis, neurogenesis, craniofacial, and skeletal development; however, only a few direct molecular targets of Sox11a have been identified. Here, we evaluate the knock-in efficiency of various HDR donor configurations and demonstrate the successful expression and localization of the resulting knock-in allele. Our results provide an efficient, streamlined approach to knock-in experiments in zebrafish, which will enable expansion of downstream experimental applications that have previously been difficult to perform. Moreover, the MYC-Sox11a line we have generated will allow further investigation into the function and direct targets of Sox11a.

Original languageEnglish
Pages (from-to)8-13
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume608
DOIs
StatePublished - Jun 11 2022

Bibliographical note

Funding Information:
We thank Brandi Bolton, Evelyn Turnbaugh, and Lucas Vieira Francisco for exceptional zebrafish care. We also thank members of the Morris lab for valuable input and technical assistance. This work was supported by grants from the NIH National Eye Institute (R01EY021769, to A.C.M.; F30EY031545, to L.A.K.)

Funding Information:
We thank Brandi Bolton, Evelyn Turnbaugh, and Lucas Vieira Francisco for exceptional zebrafish care. We also thank members of the Morris lab for valuable input and technical assistance. This work was supported by grants from the NIH National Eye Institute ( R01EY021769 , to A.C.M.; F30EY031545 , to L.A.K.)

Publisher Copyright:
© 2022 Elsevier Inc.

Keywords

  • CRISPR
  • Knock-in
  • Retina
  • Sox11
  • Zebrafish

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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