Article Figures & Data
Figures
- Figure 1.
FMRhs341 design and validation. A, Sequence alignmnt. Annotated comparison between Mus musculus (M.m.) and Homo sapiens (H.s.) of the region surrounding the fragile X repeat tract. The repeat tract is highlighted in orange, with the # symbol representing variable CGG repeat length sizes in the human population. Guide RNA sequences and their associated PAM (Protospacer Adjacent Motif) sequence are indicated by their position above the M.m. sequence, with the SNPs incorporated by the template to prevent recutting shown in red (note: Cas9 cuts several nucleotides before the PAM sequence). The green box denotes the coding region of exon 1. Sequences are written 5′ to 3′. B, Illustration of knock-in strategy. Embryos are injected with a mixture containing a single-stranded DNA template generated from human patient DNA, Cas9 protein, and two single-guide RNAs flanking the FMR1 CGG repeat tract. The mouse embryo DNA is cut by Cas9 and repaired through homologous recombination with the patient mutation template. The exchange is irreversible because of SNPs in the corresponding guide sequence on the human allele. Purple, 5′ UTR; blue, CGG repeat tract of mouse; red, CGG repeat tract of human expansion; green, coding region of exon 1. C, Confirmation of repeat size by gel electrophoresis. The FMR1hs341 amplicon was predicted to be 1353 bp in length: 190 bp upstream, 1023 bp repeat tract (341 * 3), and 140 bp downstream. Lane 1 provides a DNA ladder (1 kb Plus DNA Ladder; catalog #N3200L, New England BioLabs), lanes 3 and 5 were identified as wild type, lane 7 was identified as FMR1hs341. D, E, Sanger sequencing of upstream and downstream regions, respectively. Red box denotes guide RNA recognition sequence; blue box denotes PAM sequence. The upstream region shows strong incorporation of the human template including several SNPs located 14 bp before the predicted cut site. Upstream sequencing penetrated up to 59 CGG repeats. The downstream region demonstrates precise integration of the human template, and sequencing penetrated up to 74 CGG repeats. Two nucleotides were manually annotated for the downstream sequence. Off-target traces can be found in Extended Data Figure 1-1.
- Figure 2.
FMRhs341 does not exhibit methylation. Bisulfite sequencing of male mouse DNA. Each row represents a single animal, and each circle represents one of the 13 CG cytosines within the sequencing region. Open circle, unmethylated; closed circle, methylated; split circle, partial methylation across multiple reads; no circle, missing in reads. All animals are referenced by F<litter>.<pup>. A, Bisulfite sequencing of wild-type littermates. B, Bisulfite sequencing of Fmr1hs341 littermates. C, Bisulfite sequencing of unrelated C57BL/6 mice. D, Bisulfite sequencing of artificially methylated C57BL/6 mice, which served as a positive control that our methodology could detect methylated cytosines. Sequence traces can be found in Extended Data Figure 2-1.
- Figure 3.
FMRhs341 exhibits premutation molecular pathologies. All animals are referenced by F<litter>.<pup>. A, qRT-PCR quantification. Fmr1 mRNA is consistently higher in Fmr1hs341 male mice (red) compared with their wild-type (blue) male sibling counterparts. Values are normalized to Gapdh and Actin expression across each brain region and presented as the mean ± SEM. fCORTEX, Frontal cortex; pCORTEX, posterior cortex. B, Western blot quantification. FMRP is drastically reduced in Fmr1hs341 (red) mice compared with wild-type (blue) siblings. Values are normalized to wild-type GAPDPH expression levels.
Tables
- Table 1
Off-Target primers: primers for amplifying and sequencing candidate off-target sites
Off-target
siteGuide Chromosome Gene Mismatch Forward primer Reverse primer Sequencing primer 1 Upstream 9 Trank1 3 AGGGCCCTTAGCATTTTTGC AGCCAGCTGCAAGGAAACTC GGATGTCTCCCTCTATGCAGTG 2 Upstream 7 3 GTACTGTCTTGGTGAGGGATTG CCCAACAGAAGGCAGAGTAAG CTCATACCATACCGTGCAG 3 Upstream 1 3 CAGAGGCAGGTGGATTTCTAAG CGGTTCTAGGATTGCTGTTCTC TTCTCCATCACCATGCTGTG 4 Upstream 18 3 AGTTGCATCTCACAGTTCCTATC CTATGGGCCGGCTTCTATTATG GTAAGCTGATCTCTCCGATC 5 Upstream 12 3 GAATACAGAGCTGAGGGAATGG GGAGAACATGAGAGCTGGATATG CAGTAGTAGAGCCATCCTTAC 6 Upstream 17 3 GATTATCAGCAGGGCTAGGATG AGAGAGCATTGTGGGAATGAG CCTTTAGCTCTGGGAGGTTTC 7 Upstream 5 3 GGTTCAGTTGCTTCCCAGTT CCCGAAAGCTTGATCGAAGAG CTGAGGATCTAGAAGCACTG 8 Upstream 7 3 TTCTGTACGGTTGGCTTCTTC GGTGAGTCATCTCAGCAATCTC ACTTCAGGAGTCTTCTCTC 9 Upstream 9 Mcam 3 TGAGGGTAAGGAGAGGGTAAG GTACCATAGGACTTGAGGAATGG GTCGCTTGCTCTTACACAG 10 Upstream 4 Ctnnbip1 4 TGCCTCAGCCGGAAATAAG ACACAGACACACAGACACATAG CTAGGGTCTCAAGCCTTC 11 Downstream 7 3 CCACCTTACACTAGCCATGAAC CAAGGGCAGGATTGGAAGATAC AGAGACACAGAGGGACAAG 12 Downstream 7 3 CCACCTTACACTAGCCATGAAC CAAGGGCAGGATTGGAAGATAC AGAGACACAGAGGGACAAG Experiment Data structure Type of test 95% CI a Rotarod day 1 Small N (nonparametric) Kruskal–Wallis −66.7, 34.0 b Rotarod day 2 Small N (nonparametric) Kruskal–Wallis −40.0, 75.7 c Rotarod day 3 Small N (nonparametric) Kruskal–Wallis −23.0, 90.0 d Open field distance Small N (nonparametric) Kruskal–Wallis −1364.0, 962.0 e Zero plus distance Small N (nonparametric) Kruskal–Wallis −1010.2, 1600.7 Graphical representations of results can be found in Extended Data Table 2-1.
Figure 1-1
Off-target sequencing. A–K. Sequence traces of candidate off-target sites 1-12, respectively (note: off-target sites 11 and 12 are both found in K). Matching off-target sequence is highlighted in blue. The Fmr1hs341 F1 generation displays no sign of any off-target cutting. Download Figure 1-1, TIF file.
Figure 2-1
Bisulfite sequencing traces. Representative sequence traces of bisulfite analysis. Sequences are aligned to the wild-type sequence where potentially protected cytosines are denoted with an uppercase C. A, Wild-type sibling F88.6 shows no sign of methylation. B, Fmr1hs341 F80.17 shows no sign of methylation. C, Wild-type mouse control shows no sign of methylation. D, Artificially methylated mouse DNA (2 h incubation) demonstrates successful methylation. Download Figure 2-1, TIF file.
Table 2-1
FMRhs341 does not appear to cause motor deficits. Estimation statistics for behavioral data. All mice were males between 12 and 15 months of age, and age-matched controls were used whenever wildtype siblings were not available. Rightmost data point (FM, full mutation minus NM, no mutation or ∆) represents difference in the medians for effect size. Weighted vertical line indicates 95% confidence interval. Filled curve reflects sampling-error distribution. Generated through Ho et al., 2019 (available at https://www.estimationstats.com/ at time of publication). NM (blue): wildtype; FM (orange): Fmr1hs341. A–C. Time to first fall on the rotarod for mice across three consecutive days compared to wildtype littermates (p-values for: day 1 = 0.252; day 2 = 0.594; day 3 = 0.239) [NM: N = 11; FM: N = 9]. D. Total distance traveled in the open field arena compared to wildtype littermates (p-value = 0.342) [NM: N = 11; FM: N = 9]. E. Total distance traveled in the elevated zero maze apparatus compared to wildtype littermates (p-value = 0.540) [NM: N = 10; FM: N = 5]. Download Table 2-1, TIF file.
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