Exploring the functions of nonclassical MHC class Ib genes in Xenopus laevis by the CRISPR/Cas9 system

• Conserved cluster of a large nonclassical MHC gene family in X. tropicalis & laevis.
• Retention of most clustered nonclassical MHC genes on X. laevis large chromosome 8.
• Application of the CRISPR/Cas9 system to immune gene loss-of-function in Xenopus.
• Highly specific and efficient gene disruption of Xenopus nonclassical MHC genes.
• Single and double gene knockout X. laevis were generated to facilitate future studies.

A large family of highly related and clustered Xenopus nonclassical MHC class Ib (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 Vα6-Jα1.43 invariant T cell receptor rearrangement transcripts indicated XNC10.1 loss-of-function and deficiency in Vα6-Jα1.43 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 F0 generation of X. laevis.