DeLay BD et al. (2018), Tissue-Specific Gene Inactivation in Xenopus la...

XB-ART-54323

Genetics 2018 Feb 01;2082:673-686. doi: 10.1534/genetics.117.300468.

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Tissue-Specific Gene Inactivation in Xenopus laevis: Knockout of lhx1 in the Kidney with CRISPR/Cas9.

DeLay BD, Corkins ME, Hanania HL, Salanga M, Deng JM, Sudou N, Taira M, Horb ME, Miller RK.

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Studying genes involved in organogenesis is often difficult because many of these genes are also essential for early development. The allotetraploid frog, Xenopus laevis, is commonly used to study developmental processes, but because of the presence of two homeologs for many genes, it has been difficult to use as a genetic model. Few studies have successfully used CRISPR in amphibians, and currently there is no tissue-targeted knockout strategy described in Xenopus The goal of this study is to determine whether CRISPR/Cas9-mediated gene knockout can be targeted to the Xenopus kidney without perturbing essential early gene function. We demonstrate that targeting CRISPR gene editing to the kidney and the eye of F0 embryos is feasible. Our study shows that knockout of both homeologs of lhx1 results in the disruption of kidney development and function but does not lead to early developmental defects. Therefore, targeting of CRISPR to the kidney may not be necessary to bypass the early developmental defects reported upon disruption of Lhx1 protein expression or function by morpholinos, antisense RNA, or dominant negative constructs. We also establish a control for CRISPR in Xenopus by editing a gene (slc45a2) that when knocked out results in albinism without altering kidney development. This study establishes the feasibility of tissue-specific gene knockout in Xenopus, providing a cost-effective and efficient method for assessing the roles of genes implicated in developmental abnormalities that is amenable to high-throughput gene or drug screening techniques.

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Species referenced: Xenopus laevis
Genes referenced: atp1a1 hnf1b lhx1 slc45a2
???displayArticle.antibodies??? Kidney Ab1 Kidney Ab2
gRNAs referenced: lhx1 gRNA1 lhx1 gRNA2 lhx1 gRNA3 slc45a2 gRNA1

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	Figure 1 sgRNA targeting slc45a2 efficiently edits Xenopus embryo DNA. All data shown are from stage 10–12 embryos injected with slc45a2 sgRNA and Cas9 protein at the 1-cell stage. (A) Diagram of Slc45a2 protein showing the 12 transmembrane domains, with the region where the slc45a2 sgRNA binds outlined in black. (B) Percent of sequenced slc45a2 DNA containing different insertions and deletions. Bars shown are the mean of the percent of indel sequences from five individual embryos, error bars represent SEM. (C) Percent of indels that lead to in-frame or out-of-frame mutations. Data shown are the mean of the sequencing data from five individual embryos. (D–F) Results from a single representative embryo shown. (D) Sequencing chromatogram shows DNA editing after injection of slc45a2 sgRNA and Cas9 protein. Underlined region indicates the sgRNA binding sequence. Arrow indicates the start of degradation of the sequence due to CRISPR editing. (E) TIDE analysis shows degradation of the sequence trace in the slc45a2 CRISPR embryo after the expected Cas9 editing site. * P < 0.001 as identified by TIDE. (F) TIDE analysis prediction of the indels present in the single embryo, indicating that 3-, 12-, and 13-bp deletions are the most common indels in this embryo.
	Figure 1 sgRNA targeting slc45a2 efficiently edits Xenopus embryo DNA. All data shown are from stage 10–12 embryos injected with slc45a2 sgRNA and Cas9 protein at the 1-cell stage. (A) Diagram of Slc45a2 protein showing the 12 transmembrane domains, with the region where the slc45a2 sgRNA binds outlined in black. (B) Percent of sequenced slc45a2 DNA containing different insertions and deletions. Bars shown are the mean of the percent of indel sequences from five individual embryos, error bars represent SEM. (C) Percent of indels that lead to in-frame or out-of-frame mutations. Data shown are the mean of the sequencing data from five individual embryos. (D–F) Results from a single representative embryo shown. (D) Sequencing chromatogram shows DNA editing after injection of slc45a2 sgRNA and Cas9 protein. Underlined region indicates the sgRNA binding sequence. Arrow indicates the start of degradation of the sequence due to CRISPR editing. (E) TIDE analysis shows degradation of the sequence trace in the slc45a2 CRISPR embryo after the expected Cas9 editing site. * P < 0.001 as identified by TIDE. (F) TIDE analysis prediction of the indels present in the single embryo, indicating that 3-, 12-, and 13-bp deletions are the most common indels in this embryo.
	Figure 3 Comparison of the editing efficiency of three different sgRNAs designed to edit lhx1. (A) Diagram of the domains present in Lhx1 protein. Regions corresponding to the three lhx1 sgRNAs used in this study are represented by the black bars above the protein domains. Each sgRNA was complementary to both lhx1.L and lhx1.S. (B, C, F, G, J, and K) TIDE analysis results from the long chromosome, with pooled results from five embryos reported. (D, E, H, I, L, and M) TIDE analysis results from the short chromosome, with pooled results from five embryos reported. (B, D, F, H, J, and L) Percentage of DNA editing contributed to insertions and deletions of different sizes. Error bars represent the SEM. (C, E, G, I, K, and M) Percent of in-frame and out-of-frame insertions and deletions.
	Figure 4 Comparison of the editing efficiency of lhx1 exon 3 sgRNA in a single embryo. Results shown in A–C and D–F are from the same embryo. (A–C) Editing efficiency in lhx1.L. (D–F) Editing efficiency in lhx1.S. (A and D) Sequencing chromatogram showing degradation of the sequence in the region of the sgRNA binding site. Underlined region corresponds to the sgRNA binding site, and the black arrow marks the point of sequence degradation in the lhx1 knockout embryo. (B and E) TIDE analysis results from a single embryo showing percentage of DNA editing due to insertions and deletions of different sizes. * P < 0.001 as identified by TIDE. (C and F) Percent of in-frame and out-of-frame insertions and deletions.
	Figure 5 Knockout of lhx1 leads to kidney developmental defects. (A) Scoring system used to assess the phenotypic severity of lhx1 knockout embryos. Mild: decrease in kidney tubulogenesis in comparison to the uninjected side of the embryo; moderate: portions of the kidney tubules missing; severe: kidney tubules absent. Embryos stained with antibodies 3G8 (to label proximal tubule lumen) and 4A6 (to label the cell membranes of the intermediate, distal, and connecting tubules). (B) Targeting CRISPR knockout of lhx1 to the kidney does not decrease embryo mortality. Embryos injected at the 1-, 2- and 8-cell (blastomere V2) stages. Exogastrulation: embryos die during gastrulation; death: embryos survive gastrulation but die prior to stage 40; mistargeted: tracer not present in the kidney. (C) lhx1 targeted knockout reduces late markers of kidney development. Embryos assessed at stage 39–41 using immunostaining with 3G8 and 4A6 antibodies. (A and C) * denotes injected side of embryo. White bar, 100 μm.
	Figure 6 Knockout of lhx1 leads to a decrease in kidney developmental markers as seen by in situ hybridization. (A) Representative embryos showing that knockout of lhx1 leads to a decrease in hnf1β and atp1a1 expression in the pronephros, while slc45a2 knockout does not cause a decrease in these markers of kidney development. (B) lhx1 knockout in 1-cell embryos leads to a decrease in hnf1β and atp1a1 in comparison to slc45a2 knockout control embryos. (C) lhx1 knockout in 2-cell embryos leads to a decrease in hnf1β and atp1a1 in comparison to slc45a2 knockout control embryos. (D) lhx1 knockout in 8-cell embryos leads to a decrease in hnf1β and atp1a1 in comparison to slc45a2 knockout control embryos. I, injected side of embryo; U, uninjected side of embryo.
	Figure 7 Lhx1 protein and lhx1 RNA levels are decreased upon CRISPR knockout. (A) Immunoblot (IB) showing that knockout of lhx1 leads to a decrease in Lhx1 protein in comparison to slc45a2 knockout controls as early as embryonic stage 10â€“12. One-cell embryos were injected with 1 ng Cas9 protein and 500 pg of either slc45a2 or lhx1 sgRNA. (B) Immunoblot of embryo lysates from different stages of Xenopus development, ranging from 1-cell embryo (stage 1) to tadpole (stage 43). Levels of Lhx1 and GAPDH (loading control) protein show that Lhx1 is present in embryos throughout pronephric development (stages 12.5â€“43). (C) lhx1 in situ of stage 20 embryos shows loss of kidney staining on the injected side (white arrowheads) of lhx1 CRISPR embryos, but not in slc45a2 CRISPR embryos. No decrease in neural staining in the lhx1 knockout embryos was observed in comparison to the slc45a2 controls (black arrowheads). CRISPR knockout done in one cell of 2-cell embryos. White bar, 200 Âµm. Graph depicts severity of lhx1 loss on the injected side of the embryo. None: no loss of lhx1 staining; mild: decrease in lhx1 staining; moderate: patchy loss of lhx1 staining; severe: complete loss of lhx1 staining. (D) lhx1 immunostaining of stage 32 embryos shows loss of kidney staining on the injected side of lhx1 CRISPR embryos, but not in slc45a2 CRISPR embryos. CRISPR knockout done in one cell of 2-cell embryos. The epidermis of the embryo was removed prior to imaging. White bar, 100 Âµm. Graph depicts severity of lhx1 immunostaining loss on the injected side of the embryo. None: no loss of lhx1 staining; mild: decrease in lhx1 staining; moderate: patchy loss of lhx1 staining; severe: complete loss of lhx1 staining.
	Figure 8 Knockout of lhx1 leads to edema formation. Embryos were injected into both cells of 2-cell embryos with 1 ng Cas9 protein and 500 pg of either slc45a2 or lhx1 sgRNA, and reared to stage 43–46 for immunostaining and scoring. Error bars represent the SEM. (A) Knockout of lhx1 leads to edema formation in Xenopus embryos. (B) The percent of lhx1 knockout embryos displaying edema at stage 43–46 is higher than slc45a2 control embryos. (C) Percent of embryos with moderate (missing parts) and severe (absent) kidney phenotypes. Most lhx1 knockout embryos with edema display moderate/severe kidney phenotypes in both kidneys, while most embryos without edema have at least one normal kidney.
	Figure S1. sgRNA targeting slc45a2 efficiently edits Xenopus embryo DNA, resulting in mostly in-frame deletions. All data shown is from stage 10-12 embryos injected with slc45a2 sgRNA and Cas9 protein at the 1-cell stage. A,D,G,J,M) TIDE analysis readout showing the difference in aberrant sequences between an uninjected control embryo and an slc45a2 knockout embryo. The percent of aberrant sequences in the knockout embryo increases in comparison to the control embryo near the expected Cas9 cut site. B,E,H,K,N) slc45a2 editing efficiency in single embryos as measured by TIDE. C,F,I,L,O) Chart showing the percent of sequences that result in in frame and out of frame insertion and deletion events. * indicates p< 0.001 as reported by the TIDE analysis software.
	Figure S2.DNA editing by different lhx1 sgRNAs and Cas9 protein. All data shown is from stage 10-12 embryos injected with lhx1 sgRNA and Cas9 protein at the 1-cell stage. A,B,C) DNA editing results from a single representative embryo using a sgRNA against exon 1 of lhx1. D,E,F) DNA editing results from a single representative embryo using a sgRNA against exon 2 of lhx1. G,H,I) DNA editing results from a single representative embryo using a sgRNA against exon 3 of lhx1. A,D,E) TIDE analysis graph showing the showing the difference in aberrant sequences between an uninjected control embryo and an slc45a2 knockout embryo. The percent of aberrant sequences in the knockout embryo increases in comparison to the control embryo near the expected Cas9 cut site only for the lhx1 exon 3 sgRNA. B,E,H) lhx1 editing efficiency in single embryos as measured by TIDE. lhx1 exon 3 sgRNA is the most efficient of the three sgRNAs tested. C,F,I) Chart showing the percent of sequences that result in in frame and out of frame insertion and deletion events. lhx1 exon 3 sgRNA results in mostly out of frame deletions, while the sgRNAs against exons 1 and 2 result in mostly unedited DNA.
	Figure S3.Phenotype severity range seen using Lhx1 antibody to assess kidney development of lhx1knockout embryos. Range of phenotypes seen when lhx1is knocked out using CRISPR. Normal= no loss of Lhx1 staining, mild = reduction in Lhx1 staining, moderate = patchy loss of Lhx1 staining, severe = complete loss of Lhx1 staining. White line is 100 μm.
	Figure S4. Western blot showing loss of Lhx1 upon CRISPR knockout. Complete blot from Figure 7A, showing that Lhx1 protein levels are reduced in lhx1-knockout embryos, but not in slc45a2 knockout controls. The blot membrane was cut just above the 43 kDa marker, and the top of the blot was probed using an anti-rabbit Lhx1 antibody, while the bottom was probed using an anti-rabbit GAPDH antibody as a loading control.

References [+] :

Aslan, High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus. 2017, Pubmed, Xenbase