Banach M et al. (2017), Exploring the functions of nonclassical MHC cla...

XB-ART-52242

Dev Biol 2017 Jun 15;4262:261-269. doi: 10.1016/j.ydbio.2016.05.023.

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

Banach M , Edholm ES , Robert J .

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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.

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Species referenced: Xenopus laevis
Genes referenced: mhc1-uba1 mhc1-uba10 mhc1-uba11 mhc1-uba14 mhc1-uba6 mhc1-uba6.4 myh6 pam

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	Fig. 1. Genomic organization of X. laevis nonclassical MHC (XNC) genes and the CRISPR/Cas9 targeting strategy. (A) Genomic organization of 21 XNC genes according to the version 9.1 assembly, August 2015. Seventeen XNC genes are located on chromosome 8L within a region of 700 kb (drawn to scale). XNC2, XNC14 genes and XNC6.4 pseudogene (indicated by *) are located on the chromosome 8S. Chromosome attribution of the orphan scaffold 168 containing XNC1 is still missing. Transcriptional orientation of XNC genes is indicated by arrows. Chromosome number and gene location are based on the UCSC Genome Browser hosted at Francis Crick Institute (B) Out of 21 XNC genes XNC1, XNC11, and XNC10.1 were targeted by the CRISPR/Cas9 system in order to generate knockout transgenic X. laevis. The sgRNAs were designed against Î±1 or Î±2 domain of the targeted genes. The targeted regions are underlined, follow GG-n(18-20) schema, and have a required PAM sequence. The sgRNAs were designed with the aid of the online tools (CRISPRdirect: https://crispr.dbcls.jp/ and Zifit: http://zifit.partners.org/ZiFiT/).
	Fig. 2. XNC10.1 loss-of-function by CRISPR/Cas9-mediated editing of XNC10.1 Î±2 domain. (A) Multiple alignments of a portion of the XNC10.1 Î±2 domain targeted by the Cas9 protein and sgRNA from 8 different inbred J tadpoles (j1 to j10) with a coverage 3 or 5 cloned PCR product sequenced per tadpole (38 total number of sequences). The sequences were aligned using BioEdit Sequence Alignment software and compared to the wild type (WT) sequence of the CRISPR/Cas9 targeted region (underlined). Deletions are indicated by a (-) and insertions and nucleotide substitution as are shown in lower case letters. Note: all sequences obtained from tadpoles j2 (5 out of 5) and j9 (3 out of 3) were identical to WT. (B) Pie chart showing the relative contributions of the different types of mutations observed among the 38 sequences. (C) Assessment of XNC10.1 gene expression by RT-PCR (38 cycles with primers specific for the Î±1 and Î±2 domain) on total RNA from the 10 tadpoles (j1 to j10) used in A. RT-PCR was also performed for XNC6.1 gene, not targeted by the CRISPR/Cas9 system, and for the house keeping gene GAPDH. As positive control (+) RNA from an age-matched untreated tadpole was used. (D) Detection of invariant VÎ±6-JÎ±1.43 transcripts by qRT-PCR in an age-matched untreated control tadpole (WT) and the 8 different CRISPR/Cas9 treated tadpoles.
	Fig. 3. Assessment of off-target effects by the CRISPR/Cas9 system targeting the XNC10.1 Î±2 domain. (A) Multiple alignments of XNC Î±2 domain sequences ranked from the lowest to the highest sequence mismatches at the 3â€² end of the CRISPR/Cas9 targeted sequence. (B) Absence of mutation in the Î±2 regions of XNC6.2 and XNC8.2 (lowest mismatch with the 3â€² end targeted by the designed CRISPR/Cas9 system), XNC6.1 and XNC11 (highest mismatch with the 3â€² end). The sequences were aligned using BioEdit Sequence Alignment software and compared to the WT sequences of a region targeted by the CRISPR/Cas9 (underlined). The numbers of sequences identical to WT per total sequence analyzed are indicated in parentheses.
	Fig. 4. Generation of the XNC1 knockouts. (A) Survival of outbred (ob) developing embryos that were injected with an sgRNA targeting XNC1 and Cas9 protein. Survival and developmental stages were monitored for 50 days. Control (diamond); XNC1 knockout (black square). Statistical significance between controls and knockouts was determined using a Log-rank (Mantel-Cox) test; p<0.001. The fraction of surviving tadpoles at the end of the experiment is indicated in the parentheses. (B) Multiple alignments of the portion of the XNC1 Î±1 domain targeted by the Cas9 protein and sgRNA from 4 different tadpoles (ob 1, 4, 5 and 6) with a coverage of 4â€“19 cloned PCR product sequenced per tadpole. The sequences were aligned using BioEdit Sequence Alignment software and compared to the WT sequence of the region targeted by the CRISPR/Cas9 (underlined). Deletions are indicated by a (-) and insertions are shown in lower case letters XNC1 Î±1 domain was mutated with the majority of deletions.
	Fig. 5. Generation of the double knockout of XNC10.1 and XNC11 by the CRISPR/Cas9 system. (A) The survival of XNC10.1 and XNC11 double knockouts was monitored for 30 days: Dejellied control (black diamonds); XNC10 batch 1 (black squares); XNC10 batch 2 (black triangle); and XNC10 batch 3 (black circles). Survival was statistically different between the control and each XNC10 batch (p <0.003) but not among the 3 batches as determined by a Log-rank (Mantel-Cox) test. (B) Nine outbred (ob) tadpoles treated with the Cas9 protein and sgRNA against the Î±2 domain XNC10.1 and the Î±1 domain of XNC11 were sequenced, with a coverage 3â€“9 (XNC10.1) and 1â€“7 (XNC11) cloned PCR product sequenced per tadpole. The sequences were aligned using BioEdit Sequence Alignment software and compared to the WT sequence of the region targeted by the CRISPR/Cas9 (underlined). Both the XNC10.1 Î±2 and XNC11 Î±1 domains were mutated with the majority of detected mutations being deletions. Mosaicism was detected within each tadpole.

References [+] :

Adams, The adaptable major histocompatibility complex (MHC) fold: structure and function of nonclassical and MHC class I-like molecules. 2013, Pubmed