Immune Gene Knockout by CRISPR/Cas9
Figure 1. Thymus aplasia in foxn1 crispant F0 tadpoles. Upper panel: Thymus (arrows) located in the anterior head portion of a 3-weeks old wild type tadpole visualized under a stereomicroscope. Lower panel: Representative F0 foxn1 crispant tadpoles of matched age with barely detectable thymus (arrows).
We have adapted the CRISPR/Cas9 system to knockout important immune genes in X. laevis and investigate the roles of these genes in the development of the immune system as well as in immune homeostasis and immune responses to pathogens.
We have established a suitable experimental pipeline (Table 1) to select efficient sgRNAs and produce F0 transgenic animals for phenotypic analysis. The selected sgRNAs targeting specific exons are co-injected with Cas9 in 1-cell stage embryos. Usually, NHEJ repair pathway prevails and generated mutated progenies (F0 crispants). Genotypes of transgenic animals are determined by PCR and Sanger sequencing, while phenotypes are analysed by RT-qPCR, histology and flow cytometry. Importantly, as shown in Figure 1, CRISPR/Cas9-mediated gene KO in F0 animals is efficient despite being mosiac and produces detectable functional phenotypes useful for immunological studies.
Thus, we can use a fraction of F0 Tg animals to characterize mutation defects, while keeping another fraction of animals for breeding and selecting F1,2 mutant lines.
Experimental pipeline for generating immune-deficient transgenic lines
| 1 |
Design 2-3 sgRNAs targeting a specific exon |
| 2 |
Co-injected of each sgRNA with Cas9 in 1-cell stage embryos |
| 3 |
RNA extraction of embryos 48 hr post-injection |
| 4 |
Mutation efficiency analysis by PCR and Sanger sequencing |
| 5 |
Transgenesis with the most efficient sgRNA |
| 6 |
Phenotypic analysis of F0 transgenic tadpole by transcriptomics and flow cytometry |
| 7 |
Breeder selection for F1 and F2 generation |
The F0 Transgenic Lines in Our Unit
| Gene |
Deficiency |
F0 Mutations (%KO Score or % Indel) |
| foxn1 |
Thymus development |
34.2% or 37.3% |
| rag2 |
B and T cells |
57% or 54.3% |
| trac |
Mature T cells |
39.57% or 31.29% |
| foxp3 |
Regulatory T cells |
75.43% or 69% |
| ifn1 |
Innate immune responses |
28% or 20.67% |
| tap2 |
Antigen presentation |
72.86% or 62.43% |