Journal
DEVELOPMENTAL BIOLOGY
Volume 426, Issue 2, Pages 261-269Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ydbio.2016.05.023
Keywords
Gene knockout; Reverse genetic; Genomics; Amphibians; Comparative immunology; XNC
Categories
Funding
- National Institute of Allergy and Infectious Diseases (NIH/NIAID) [R24-AI-059830]
- Ruth L. Kirschstein Predoctoral F31 from the National Cancer Institute (NIH/NCI) [F31CA192664]
- NIH-T32 training grant [AI007285]
- Nation Science Foundation [IOS-1456213]
- Direct For Biological Sciences
- Division Of Integrative Organismal Systems [1456213] Funding Source: National Science Foundation
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A large family of highly related and clustered Xenopus nonclassical MHC class lb (XNC) genes influences Xenopus laevis immunity and potentially other physiological functions. Using RNA interference (RNAi) technology, we previously demonstrated that one of XNC genes, XNC10.1, is critical for the development and function of a specialized innate T (iT) cell population. However, RNAi limitation such as a variable and unstable degree of gene silencing in F0 and F1 generations is hampering a thorough functional analysis of XNC10.1 and other XNC genes. To overcome this obstacle, we adapted the CRISPR/Cas9-mediated gene editing technique for XNC genes. We efficiently and specifically generated single gene knockouts of XNC10.1, XNC11, and XNC1 as well as double gene knockouts of XNC10.1 and XNC11 in X. laevis. In single XNC10.1 knockout X laevis tadpoles, the absence of XNC10.1 and V alpha 6-J alpha 1.43 invariant T cell receptor rearrangement transcripts indicated XNC10.1 loss-of-function and deficiency in Va6-Ja1.43 iT cells. Notably, targeting XNC10.1 did not affect neighboring XNC genes exhibiting high sequence similarity. Furthermore, XNC1 gene disruption induced mortality during developmental stage 47, suggesting some non-immune but essential function of this gene. These data demonstrate that the CRISPR/Cas9 system can be successfully adapted for genetic analysis in FO generation of X laevis. (C) 2016 Elsevier Inc. All rights reserved.
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