Lineage tracing of genome-edited alleles reveals high fidelity axolotl limb regeneration
- Yale University, United States
Figures
Figure 1 with 3 supplements
Comparison of CRISPR-generated alleles in original and regenerated axolotl limbs.
(A) Schematic of alleles generated by CRISPR-induced mutagenesis in original and regenerated axolotl limbs. Axolotls were zygotically injected with cas9 mRNA and single gRNA (top left), producing …
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Figure 1—source data 1
All allele counts in all animals.
- https://doi.org/10.7554/eLife.25726.008
Figure 1—figure supplement 1
The log scores of reads per million (RPM) of every allele in the original limb compared with those of the secondary limb for each individual animal used in this study.
https://doi.org/10.7554/eLife.25726.003
Figure 1—figure supplement 2
Characterization of the frequency with which alleles co-occur in different animals.
(A) Distribution of unique and shared indel sequences across animals injected with same gRNA. When individual allele sequences detected in animals injected with the same gRNAs are compared across …
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Figure 1—figure supplement 2—source data 1
Counts of alleles that are unique and shared between animals.
- https://doi.org/10.7554/eLife.25726.005
Figure 1—figure supplement 3
Distribution of unique and shared indel sequences across animals injected with reduced concentrations of cas9 and gRNAs.
Individual embryos were injected with 5 pg of each of 5 guide RNAs and either 100 or 1000 pg of cas9 mRNA. NGS of amplified targets from genomic DNA extracted two weeks post-injection from entire …
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Figure 1—figure supplement 3—source data 1
Counts of all alleles in animals injected with varying concentrations of cas9 and gRNAs.
- https://doi.org/10.7554/eLife.25726.007
Figure 2
The sizes and fold changes of all alleles found in original and regenerated limbs in this study.
(A) Pie chart depicting numbers of all alleles identified in original limbs binned by RPM. The majority of alleles detected are low frequency (i.e., represent less than 1.6% of all sequence reads in …
Figure 3
Comparison of all alleles in primary and secondary limb in one representative animal.
(A) Depiction of all identified alleles and their relative sizes in primary and secondary limbs of one individual mutagenized with meth t2 gRNA. The sequence of the gRNA and protospacer adapter …
Figure 4 with 2 supplements
Comparison of alleles detected in primary, secondary, and tertiary limbs.
(A) Schematic depicting quantification of alleles in primary, secondary, and tertiary limbs. Mutagenized primary limbs were amputated, allowed to fully regenerate to produce secondary limbs, again …
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Figure 4—source data 1
Mutation frequency and allelic change in all animals.
- https://doi.org/10.7554/eLife.25726.014
Figure 4—figure supplement 1
Spontaneous polydactyly in animal exhibiting dissimilar primary and secondary limb allelic identities.
(A) Original amputated limb of animal in which egfp has been targeted (EGFP 2) revealing normal development at time of amputation (green channel only). (B) Secondary limb of same animal depicted in A…
Figure 4—figure supplement 2
The log scores of reads per million (RPM) of every allele in the original limb compared with those of the tertiary limb for each individual animal that underwent secondary amputations.
https://doi.org/10.7554/eLife.25726.013
Figure 5 with 2 supplements
Allelic compositions of tissue-enriched samples vary relative to each other.
The heat map of the r2 log scores of normalized RPM +1 values for alleles of all tissue-enriched samples from four quaternary limbs and hands. More isolatable samples (nerve, vessel, and blood) …
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Figure 5—source data 1
All allele counts in all tissues.
- https://doi.org/10.7554/eLife.25726.018
Figure 5—figure supplement 1
Identification and quantification of tissue-enriched alleles.
(A) Log scores of RPM of 171 alleles detected in tissue-enriched samples and hands from four quaternary limbs. Each column represents one allele across 6 tissue-enriched samples and hand. Alleles …
Figure 5—figure supplement 2
Alleles are disproportionally lost after first amputation.
36/54 alleles detected in the primary limb but not in the secondary, were also detected in the tertiary limb or tissue-enriched samples from the quaternary limb. 5/5 alleles present in the secondary …
Additional files
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Supplementary file 1
Primers and gRNA target sequences.
- https://doi.org/10.7554/eLife.25726.019
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Transparent reporting form
- https://doi.org/10.7554/eLife.25726.020
