Multiplex CRISPR/Cas screen in regenerating haploid limbs of chimeric Axolotls
- Yale University, United States
Figures
Figure 1
Haploid-diploid chimeric generation and lineage analysis.
(A) Schematic of haploid-diploid chimera generation. Gynogenetic haploids are generated by in vitro activation of unfertilized eggs with UV-enucleated sperm and subsequently mutagenized using …
Figure 2 with 2 supplements
Haploid-diploid chimeric axolotl.
(A) Composite fluorescent image of a chimeric axolotl produced from a limb bud graft from an RFP+ haploid embryo to a GFP+ diploid host. Scale bar = 1 cm. (B) Composite fluorescent image of haploid …
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Figure 2—source data 1
The number of diploid white to diploid GFP+ grafts that were performed to determine the optimal embryonic stage for limb bud grafting.
- https://cdn.elifesciences.org/articles/48511/elife-48511-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Characterization of haploid larvae.
(A) Fluorescent image of a chromosome squash of a diploid cell (2n = 28). A’ and A’’ Fluorescent images of chromosome squashes from two haploid cells (1n = 14). Chromosomes were stained with Hoescht …
Figure 2—figure supplement 2
Time course of haploid and diploid limb regeneration.
Four diploid limbs and four haploid limbs of eight stage-matched animals were amputated and imaged on days 0, 3, 7, 10, 16, 23, 30, 37. Haploids (top two rows) regenerate complete limbs and retain …
Figure 3 with 2 supplements
Control alleles.
(A) Comparison of all alleles generated in the controls (methyltransferase plus tyrosinase) in the original and regenerated haploid limbs of 12 animals. The log scores of the reads per ten thousand …
Figure 3—figure supplement 1
Comparison of all alleles generated in the controls (methyltransferase and tyrosinase) in the original and regenerated haploid limbs of 12 animals shown individually and compared to the entire remaining set of control alleles.
In each graph, the open red circles depicts the log scores of the RP10K of every allele in the original limb compared to that of the same allele in the regenerated limb of an individual animal, …
Figure 3—figure supplement 2
Histograms depicting the log of fold change after regeneration for alleles detected in the controls (methyltransferase-like and tyrosinase).
(A) All alleles detected in the controls. (B) All alleles that occur with a mutation frequency of 1.60% or less. The majority of these alleles undergo less than a two-fold change after regeneration. …
Figure 4 with 1 supplement
Fetuin-b alleles compared to all other target gene and control alleles.
(A) Linear regression plot of the log2(RP10K) score for all alleles of fetuin-b detected in the first and regenerated haploid limbs of 11 animals. The log scores of alleles in the primary limb …
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Figure 4—source data 1
Raw number of reads, normalized reads, and log2(RP10K) score for all mutant alleles of every targeted gene in each mutant limb in this study.
- https://cdn.elifesciences.org/articles/48511/elife-48511-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Linear regression plot of the log2(RP10K) score for all alleles in primary and secondary of each targeted gene for which no significant deviation was detected from that of control alleles (akap8l, p=0.069; cacng, p=0.166; hnrnpa0, p=0.371; hoxa9, p=0.637; hoxb13, p=0.053; myl6, p=0.850; pmp2, p=0.624; rcc, p=0.176; tyr, p=0.532,; zic5, p=0.480; ANCOVA).
Best-fit lines are added for genes for which more than one allele was detected. Numbers of animals in which mutations were detected are listed under each gene name.
Figure 5
Catalase alleles compared to all other target gene and control alleles.
(A) Linear regression plot of the log2(RP10K) score for all alleles of catalase detected in the first and regenerated haploid limbs of three animals. The log scores of alleles in the primary limbs …
Figure 6
Larval tail regeneration in tyrosinase, catalase, and fetuin-b mutants.
(A) Regenerative outgrowth of tail in high-level tyrosinase, catalase, and fetuin-b F0 mutants. While no significant difference is detected at early time points, both fetuin-b and catalase mutants …
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Figure 6—source data 1
Regenerative outgrowth measurements and genotyping data for tyrosinase, catalase, and fetuin-b F0 mutants.
- https://cdn.elifesciences.org/articles/48511/elife-48511-fig6-data1-v1.xlsx
Tables
Table 1
The numbers of all alleles in the first limbs of controls, all targets, fetuin-b, all targets excluding fetuin-b, catalase, and all targets excluding catalase that are sorted by mutation frequency and log of fold change.
| Controls | All targets | |||||||
|---|---|---|---|---|---|---|---|---|
| Allele Frequency | Log of fold change | Allele Frequency | Log of fold change | |||||
| (Low) Frequency < 1.6% | < 2 | > 2 | (Low) Frequency < 1.6% | < 2 | > 2 | |||
| Alleles Lost | 22 | 5 | 17 | Alleles Lost | 60 | 24 | 36 | |
| Alleles Preserved | 53 | 35 | 18 | Alleles Preserved | 94 | 71 | 23 | |
| Sum | 75 | 40 | 35 | Sum | 154 | 95 | 59 | |
| Allele Frequency | Log of fold change | Allele Frequency | Log of fold change | |||||
| Frequency > 1.6% | < 2 | > 2 | Frequency > 1.6% | < 2 | > 2 | |||
| Alleles Lost | 0 | 0 | 0 | Alleles Lost | 2 | 0 | 2 | |
| Alleles Preserved | 17 | 15 | 2 | Alleles Preserved | 20 | 13 | 7 | |
| Sum | 17 | 15 | 2 | Sum | 22 | 13 | 9 | |
| Total alleles: 92 | Total alleles: 176 | |||||||
| fetuin-b | All targets except fetuin-b | |||||||
| Allele Frequency | Log of fold change | Allele Frequency | Log of fold change | |||||
| (Low) Frequency < 1.6% | < 2 | > 2 | (Low) Frequency < 1.6% | < 2 | > 2 | |||
| Alleles Lost | 20 | 9 | 11 | Alleles Lost | 40 | 15 | 25 | |
| Alleles Preserved | 25 | 21 | 4 | Alleles Preserved | 69 | 50 | 19 | |
| Sum | 45 | 30 | 15 | Sum | 109 | 65 | 44 | |
| Allele Frequency | Log of fold change | Allele Frequency | Log of fold change | |||||
| Frequency > 1.6% | < 2 | > 2 | Frequency > 1.6% | < 2 | > 2 | |||
| Alleles Lost | 2 | 0 | 2 | Alleles Lost | 0 | 0 | 0 | |
| Alleles Preserved | 1 | 0 | 1 | Alleles Preserved | 19 | 13 | 6 | |
| Sum | 3 | 0 | 3 | Sum | 19 | 13 | 6 | |
| Total alleles: 48 | Total alleles: 128 | |||||||
| catalase | All other targets except catalase | |||||||
| Allele Frequency | Log of fold change | Allele Frequency | Log of fold change | |||||
| (Low) Frequency < 1.6% | < 2 | > 2 | (Low) Frequency < 1.6% | < 2 | > 2 | |||
| Alleles Lost | 6 | 1 | 5 | Alleles Lost | 54 | 23 | 31 | |
| Alleles Preserved | 1 | 1 | 0 | Alleles Preserved | 93 | 70 | 23 | |
| Sum | 7 | 2 | 5 | Sum | 147 | 93 | 54 | |
| Allele Frequency | Log of fold change | Allele Frequency | Log of fold change | |||||
| Frequency > 1.6% | < 2 | > 2 | Frequency > 1.6% | < 2 | > 2 | |||
| Alleles Lost | 0 | 0 | 0 | Alleles Lost | 2 | 0 | 2 | |
| Alleles Preserved | 1 | 0 | 1 | Alleles Preserved | 19 | 13 | 6 | |
| Sum | 1 | 0 | 1 | Sum | 21 | 13 | 8 | |
| Total alleles: 8 | Total alleles: 168 | |||||||
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Ambystoma mexicanum) | cagg:egfp | Ambystoma Genetic Stock Center (Sobkow et al., 2006) | AGSC Cat# 110A, RRID:AGSC_110A | |
| Genetic reagent (Ambystoma mexicanum) | cagg:nls-mcherry | Ambystoma Genetic Stock Center (Kragl et al., 2009) | AGSC Cat# 112A, RRID:AGSC_112A | |
| Chemical compound, drug | MS-222 | Western Chemical | ANADA #200–226 | |
| Chemical compound, drug | Human chorionic gonadotropin (Chorulon,) | Merck Animal Health | NADA 140–927 | |
| Gene (Ambystoma mexicanum) | msx2 | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000067092 | |
| Gene (Ambystoma mexicanum) | prmt1 | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000062704 | |
| Gene (Ambystoma mexicanum) | myl6 | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000067862 | |
| Gene (Ambystoma mexicanum) | fetub | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000227254 | |
| Gene (Ambystoma mexicanum) | hoxc8 | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000065333 | |
| Gene (Ambystoma mexicanum) | akap8l | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000192860 | |
| Gene (Ambystoma mexicanum) | hrnrpa0 | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000081837 | |
| Gene (Ambystoma mexicanum) | hsd17b10 | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000257015 | |
| Gene (Ambystoma mexicanum) | hoxb9 | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000035333 | |
| Gene (Ambystoma mexicanum) | tyrosinase | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000179254 | |
| Gene (Ambystoma mexicanum) | etv4 | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000233035 | |
| Gene (Ambystoma mexicanum) | cacng1 | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000081988 | |
| Gene (Ambystoma mexicanum) | catalase | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000186723 | |
| Gene (Ambystoma mexicanum) | hoxb13 | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000007929 | |
| Gene (Ambystoma mexicanum) | zic5 | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000057641 | |
| Gene (Ambystoma mexicanum) | ecm1 | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000123229 | |
| Gene (Ambystoma mexicanum) | cornifelin | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000173184 | |
| Gene (Ambystoma mexicanum) | dsg-like | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000056512 | |
| Gene (Ambystoma mexicanum) | enpp2 | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000217071 | |
| Gene (Ambystoma mexicanum) | fabp2 | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000084459 | |
| Gene (Ambystoma mexicanum) | pmp2 | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000238807 | |
| Gene (Ambystoma mexicanum) | kcne1 | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000121776 | |
| Gene (Ambystoma mexicanum) | krt6a | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000060835 | |
| Gene (Ambystoma mexicanum) | rcc1 | Axolotl transcriptome assembly 3.4 | AMEXTC_0340000210022 | |
| Recombinant DNA reagent | MLM3613 | (Hwang et al., 2013) | RRID: Addgene plasmid 42251 | Cas9 expression vector |
| Peptide, recombinant protein | Cas9 | PNABio | Cat. #: CP04-500 | |
| Commercial assay or kit | mMessage mMachine Kit | ThermoFisher | Cat. #: Am1345 | |
| Commercial assay or kit | MAXIscript SP6/T7 Transcription Kit | ThermoFisher | Cat. #: Am1322 | |
| Chemical compound, drug | MS-222 | Sigma Aldrich | SML1656 | |
| Software, algorithm | Geneious Software | Biomatters | RRID:SCR_010519 |
Additional files
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Supplementary file 1
Primer and gRNA sequences used in study.
- https://cdn.elifesciences.org/articles/48511/elife-48511-supp1-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/48511/elife-48511-transrepform-v1.docx
