December 15, 2015;
CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease genes in Xenopus.
Congenital malformations are the major cause of infant mortality in the US and Europe. Due to rapid advances in human genomics, we can now efficiently identify sequence variants that may cause disease in these patients. However, establishing disease causality remains a challenge. Additionally, in the case of congenital heart
disease, many of the identified candidate genes are either novel to embryonic development or have no known function. Therefore, there is a pressing need to develop inexpensive and efficient technologies to screen these candidate genes for disease phenocopy in model systems and to perform functional studies to uncover their role in development. For this purpose, we sought to test F0 CRISPR based gene editing as a loss of function strategy for disease phenocopy in the frog model organism, Xenopus tropicalis. We demonstrate that the CRISPR/Cas9 system can efficiently modify both alleles in the F0 generation within a few hours post fertilization, recapitulating even early disease phenotypes that are highly similar to knockdowns from morpholino oligos (MOs) in nearly all cases tested. We find that injecting Cas9 protein is dramatically more efficacious and less toxic than cas9 mRNA. We conclude that CRISPR based F0 gene modification in X. tropicalis is efficient and cost effective and readily recapitulates disease and MO phenotypes.
Disease Ontology terms:
ALBINISM, OCULOCUTANEOUS, TYPE IA; OCA1A
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Fig. 1. Strategy for custom sgRNA design, Fragment Analysis, and study of tyrosinase CRISPR phenotype and genotype. (a) Strategy for custom sgRNA design (PCR method) with the red box outlining the forward primer for any target gene of interest. *This strategy was modified from that described by Nakayama et al. by addition of 5 bp anchor sequence and use of Ambion’s MegaShortscript kit, which greatly increases RNA yield (See Methods). The forward primer consists of 5 bp anchor sequence (red), T7 promoter (yellow), 18 bp target sequence (green), and 20 bp complimentary overhang with reverse primer (black). The stock reverse primer consists of 20 bp complementary overhang (black) and the remaining sgRNA sequence (blue). With one PCR step we generate the full sgRNA template. We then use in vitro transcription to generate the sgRNA which is co-injected with cas9 mRNA or protein. (b) Strategy for fragment analysis. Forward primer with a 5′-m13 overhang sequence is used with a R-primer to PCR amplify the target locus. A 2nd PCR step with a florescent m13 Primer and the same R-primer are used to generate a fluorescently tagged PCR amplicon, which is run on a capillary electrophoresis gel and analyzed. (c) Stage 42 embryos and their FA genotype (dorsal view, anterior to the left). FA genotype – X-axis size of amplicon in basepairs (bp), Y-axis relative intensity of PCR signal to loading control. WT tadpole and the corresponding 261 bp PCR amplicon; Tadpole injected at 1-cell stage with cas9 mRNA+tyr sgRNA and corresponding 257 bp FA peak (4 bp deletion). GFP co-expression is observed in whole embryo; Tadpole injected at 1-of-2-cell stage with cas9 mRNA+tyr sgRNA on left side (GFP co-expression only on left) and both deletion and WT peaks detected with FA; Tadpole injected with Cas9 protein and tyr sgRNA at 1-cell stage with 257 bp deletion peak on FA.
Fig. 2. Time course of CRISPR/Cas9 deletions. All embryos injected with tyr sgRNA and either injected with cas9 mRNA or Cas9 protein. X-axis=time/ stage of embryos post-injection (number of embryos in parenthesis). Y-axis=% embryos with corresponding FA genotype (blue=WT allele only, green=both alleles detected, red=mutant allele only). Statistical analysis performed by Fisher exact probability test, *=p<0.05.
Fig. 3. CRISPR injected embryos phenocopy established mutant or MO knockdown phenotypes in the F0 generation. Left panel=WT embryo, Middle panel=MO injected embryo, Right panel=CRISPRp injected embryo. (a) Foxj1 CRISPRp – Immunohistochemistry of stage 28 embryos with acetylated tubulin to mark surface cilia. (b) Pax8 CRISPRp – in situ hybridization of stage 38 embryos with pax2 probe to stain kidney tubule. Black arrow points to pronephros.
Supplemental Fig. 1. Topo-cloned sequence of deletion and insertion peaks. Green marks the target sequence,-marks deletions or insertions, * marks identical sequence to WT. FA genotype – X-axis=size of amplicon in basepairs (bp), Y-axis=relative intensity of PCR signal to loading control. (a) WT FA peak at 261 bp and corresponding sequence from n=11 clones. (b) Embryo injected with Cas9 protein+tyr sgRNA. FA shows two main peaks at 253 bp and 257 bp, corresponding to 8 bp and 4 bp deletions and their corresponding sequences from n=21 clones. (c) Embryo injected with Cas9 protein+tyr sgRNA. FA shows two main peaks at 257 bp and 266 bp, corresponding to 4 bp deletion and 5 bp insertion and their corresponding sequences from n=12 clones.
Supplemental Fig. 2. Toxicity graph comparing cas9 mRNA vs. protein. X-axis=different injection conditions, Y-axis=% embryos dead (red) or alive (green).
Supplemental Fig. 3. Examples of fragment analysis, western blot, and immunohistochemistry for various genes tested. FA genotype – X-axis=size of amplicon in basepairs (bp), Y-axis=relative intensity of PCR signal to loading control. (a–e) Fragment analysis of embryos injected with Cas9 protein+sgRNA – galnt11, dnah9, ccdc40, beta-catenin, pax8. (f) Western blot analysis using antibody against Foxj1. Quantification of changes in protein level was normalized to GAPDH and calculated using ImageJ software from NIH. (g) Immunohistochemistry of stage 28 embryos with acetylated tubulin to mark surface cilia. Left to right – WT, dnah9, ccdc40, and galnt11.