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

Abstract

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.

Distinct MHC class I-like interacting invariant T cell lineage at the forefront of mycobacterial immunity uncovered inXenopus

“…S6). The relatively high degree of mosaicism within individual F 0 tadpoles has been previously reported and is likely due to a rapid embryogenesis and continuous CRISPR/Cas9-mediated genome modifications after the one-cell embryo stage, which results in independent doublestrand breaks with different mutations upon imprecise DNA repair mechanism (22). To evaluate the potential off-target effects on Jα segments proximal to, or sharing sgRNA motifs with Jα1.14, we sequenced three Jα gene segments in the three tadpoles carrying the highest number of Jα1.14 mutations and did not find any off-target mutations (SI Appendix, Fig.…”

“…2): (i) the disruption of the Jα1.14 gene segment by the CRISPR/Cas9 genome editing system to prevent the iVα45-Jα1.14 TCR rearrangement; and (ii) I-SecI meganucleasemediated transgenesis targeting the CDR3 region to silence the Vα45-Jα1.14 rearrangement. We have shown that the CRISPR/ Cas9 system is a robust method for targeting with high specificity a single member of multigene families in X. laevis (22). Accordingly, we designed a single guide RNA (sgRNA) against the Jα1.14 gene ( Fig.…”

Tissue-Specific Gene Inactivation inXenopus laevis: Knockout oflhx1in the Kidney with CRISPR/Cas9

“…CRISPR gene editing is new in the allotetraploid X. laevis (Lane et al 2015;Wang et al 2015;Jaffe et al 2016;Banach et al 2017), and tissue-targeted CRISPR knockouts have not been reported. Our results indicate that CRISPR can efficiently target a tissue of interest.…”

Tumor Immunology Viewed from Alternative Animal Models—the Xenopus Story

“…In addition to a single classical MHC class I gene per genome, X. laevis has a large family of MHC class I-like genes ( mhc1b or previously known as XNC) , which are clustered outside the MHC locus [53, 54]. mhc1b genes encode molecules with the typical three immunoglobulin α domain structure, exhibit low polymorphism, and in general have limited tissue distribution [53, 55].…”