Generation of a specific f-Prdm16 deletion in mice which is lethal and causes a severe defect

To specifically delete f-Prdm16, we injected fertilized C57BL/6 embryos with PX330 plasmid containing gRNA sequences targeting exon 2 of Prdm16. Two chimeric mice were obtained from the litter, which

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were bred to WT C57BL/6 mice to generate heterozygotes. Sequencing of DNA from the two mice revealed two distinct indels, one 47bp long and another 13bp long (Δ47-fPrdm16+/- _{and Δ13-fPrdm16}+/-₎ (Figure 4-18A). Both of these deletions are indivisible by three and therefore frameshifting, and both lead to premature stop codons shortly after deletion (Figure 4-18B).

Figure 4-18: Generation of two mouse strains with frameshifting indels in Prdm16 exon 2. (A) Map of

the 47bp and 13bp deletions (in red) obtained from mice using CRISPR/Cas9 pronuclear injection of gRNA targeting Prdm16 exon 2. (B) Comparison of the frameshifted amino acid sequences (red) from 47bp and 13bp deletions to WT. Both frameshifts lead to premature stop codons (*) shortly after deletion.

Paired breeding of either heterozygous Δ47-fPrdm16+/- _{or Δ13-fPrdm16}+/- _{mice produced progeny with no} homozygous deleted mice, signifying that a global deletion of f-Prdm16 is a lethal mutation in mice. Therefore, as with the global Prdm16-/- _{deletion, fetal liver needed to be studied to discern hematopoietic} properties. Day (E)13.5-16.5 embryos were distributed in Mendelian ratios (Figure 4-19A). To confirm a specific loss of f-Prdm16 in these mice, we employed subtractive qPCR of fetal liver HSCs as described in Figure 4-16B. We detected a loss of f-Prdm16, but not s-Prdm16 in these cells, when calculated as either

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mRNA molecules per cell or by percentage (Figure 4-19B). We therefore concluded that the

Δ47-fPrdm16-/- _{and Δ13-fPrdm16}-/- _{frameshifting mutations result in a specific, global deletion of}

f-Prdm16, or, viewed from the opposite perspective, a “s-Prdm16-only” genotype.

Figure 4-19: Global f-Prdm16 deletion is embryonic lethal and retains expression of s-Prdm16. (A)

Mendelian distribution of E13.5-15.5 embryos of Δ47-fPrdm16 mice (n = 62). (B) Subtractive qPCR quantification of Prdm16 isoform mRNA copy number (top panel) and percent isoform expression (bottom panel) from fetal liver HSCs of Δ47-fPrdm16-/- _{(KO) and WT littermates shows selective loss of f-Prdm16 in} the mutants. (n.s = P > 0.05, Chi-square test)

Having confirmed the specificity of f-Prdm16 global deletion, we next assessed the hematopoietic phenotype of these mice, using fetal livers. In both the Δ47-fPrdm16-/- _{and Δ13-fPrdm16}-/- _{mice, the} frequency (Figure 4-20A) and absolute number (Figure 4-20B) of HSCs (Lin-_cKit+_Sca1+_CD48-_CD150+_{) was}

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however, indicating a specific depletion in HSCs without a loss of downstream progenitors (Figure 4-20C). Notably, we saw an intermediate phenotype in heterozygous Δ47-fPrdm16+/- _{and Δ13-fPrdm16}+/- _fetal liver. This is similar to what our lab previously observed in total Prdm16+/- _{fetal liver, where heterozygotes} had an intermediate phenotype between WT and homozygous KO. Because we observed an identical phenotype in both Δ47-fPrdm16-/- _{and Δ13-fPrdm16}-/- _{fetal liver, further experiments were performed} with Δ47-fPrdm16-/- _{mice. Furthermore, the strong similarity between the Δ47-fPrdm16}-/- _and

Δ13-fPrdm16-/- _{fetal liver compartments substantially reduces the chances of artifacts due to a} non-specific gRNA targeting event. Experimental WT littermate controls were also derived from CRISPR/Cas9 blastocyst injection. Therefore, any non-specific off-target deletions should be randomly distributed between WT and Δ47-fPrdm16-/- _{or Δ13-fPrdm16}-/- _{mice. Finally, the fact that SL-TSS}-/- _mice had no hematopoietic phenotype argues against the possibility of off-target effects.

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Figure 4-20: Specific loss of HSCs in f-Prdm16-/- _{fetal liver. (A) Frequency (%) and (B) absolute number of} HSCs (Lin-_cKit+_Sca1+_CD48-_CD150+_{) in fetal liver of Δ47-fPrdm16}-/- _{(KO), Δ47-fPrdm16}+/- _{(HET) and WT} littermate mice (n = 34 mice), or Δ13-fPrdm16-/- _{(KO), Δ13-fPrdm16}+/- _{(HET) and WT (n = 28 mice). (C)} Frequency of LSK cells (Lin-_Sca1+_cKit+_{) using the same fetal liver samples from (A). (P > 0.25 for all}

comparisons). (n.s = P > 0.05; * = P < 0.05; ** = P < 0.01; *** = P < 0.001, One-way ANOVA for multiple comparisons)

We then performed competitive transplants of Δ47-fPrdm16-/- _{and WT littermate fetal liver to compare} the effect of f-Prdm16 deletion on HSC maintenance. Δ47-fPrdm16-/- _{fetal liver cells showed a severe} repopulation defect, on a similar order to that observed in Prdm16-/- _{fetal liver (Figure 4-21A). However,} in stark contrast to Prdm16-/- _{competitive transplants, the few reconstituting donor Δ47-fPrdm16}-/- _cells were strongly lymphoid biased compared to repopulating WT cells, and within the lymphoid population, were further biased toward the B-cell lineage (Figure 4-21B). A comparison of proliferation and apoptosis between Δ47-fPrdm16-/- _{and WT HSCs showed no significant differences – both HSC populations had} similar levels of cycling (Figure 4-21C), as measured by Ki-67 staining, and apoptosis, measured as the percentage of HSCs expressing cleaved Caspase3 (Figure 4-21D). Furthermore, and similar to global

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Prdm16-/- _{fetal liver and Prdm16}fl/fl_{.Vav-Cre bone marrow, there was no defect in engraftment of donor} cells to the bone marrow after 24 hours in Δ47-fPrdm16-/- _{fetal liver cells (Figure 4-21E).}

Figure 4-21: Severe HSC reconstitution defect and B-cell bias in Δ47-fPrdm16-/- _{HSCs. (A) Peripheral blood} donor chimerism of transplanted Δ47-fPrdm16-/- _{(KO) and WT littermate fetal liver HSCs in competitive} transplants with CD45.1 bone marrow, measured 16-weeks post-transplant (n = 12-14 mice, 3 independent transplants). (B) Percent of donor cells from (A) that were lymphoid (CD19+ _{or CD3}+_{) and the}

percentage of those that were B-cells (CD19+_{) (n = 12-14). (C) Percent KI-67}+ _{and (D) cleaved Caspase3}+ _FL

HSCs in Δ47-fPrdm16-/- _{and WT littermate embryos(n = 3). (E) Percent donor CD45.2 cells in bone marrow} of recipient mice 24 hours after transplantation of fetal liver cells from either Δ47-fPrdm16 (KO) or WT littermates. (n = 5 recipient mice). (n.s = P > 0.05; * = P < 0.05; ** = P < 0.01; *** = P < 0.001, One-way ANOVA for multiple comparisons or Student’s t-test for single comparisons).

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Repopulating fetal liver HSCs are lymphoid-biased compared to adult HSCs, but even taking this into account, Δ47-fPrdm16-/- _{repopulation was exceptionally lymphoid-biased – almost entirely comprised of} lymphoid cells, particularly CD19+ _{B-cells. Our results demonstrate that the f-Prdm16 isoform is essential}

to HSC maintenance as deletion results in a defect in competitive transplantation similar to the

Prdm16-/- _{and Prdm16}fl/fl_.Vav-Cre+ _{deletions. The Δ47-fPrdm16}-/- _{mouse, however, can conversely be} thought of as a “s-Prdm16 only” mouse, and the fact that there is a strong lymphoid bias among the few reconstituting cells suggest that s-Prdm16 supports limited lymphoid reconstitution, but is not sufficient for the effective maintenance of HSCs.

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