Mike Gallagher
@geneticsmike7.bsky.social
Geneticist and stem cell biologist studying epigenetics and neurological disease at Whitehead Institute and MIT.
19/ strangely, leaky SALL1 is not due to unwanted splicing in the absence of drug, but rather the unspliced pre-mRNA somehow escapes the nucleus and is translated in-frame to make the full-length protein. We still don't know how this works.
November 2, 2025 at 2:35 PM
19/ strangely, leaky SALL1 is not due to unwanted splicing in the absence of drug, but rather the unspliced pre-mRNA somehow escapes the nucleus and is translated in-frame to make the full-length protein. We still don't know how this works.
17/ we also show that we can get inducible gene editing with Xon hPSCs, but only if sgRNAs are introduced transiently (panels C-E). Cas9 must have enough leakiness for maximal editing with constitutive lentiviral sgRNA introduced into hPSCs (A-B)
November 2, 2025 at 2:32 PM
17/ we also show that we can get inducible gene editing with Xon hPSCs, but only if sgRNAs are introduced transiently (panels C-E). Cas9 must have enough leakiness for maximal editing with constitutive lentiviral sgRNA introduced into hPSCs (A-B)
16/ what about other genes? Here we show Xon works for inducible control of GRN expression, and we can overexpress GRN in iPSC-derived microglia, which already express boatloads of it. P2A peptides improve both GRN and SMAD, and importantly reduce N-terminal tag to a single aa.
November 2, 2025 at 2:31 PM
16/ what about other genes? Here we show Xon works for inducible control of GRN expression, and we can overexpress GRN in iPSC-derived microglia, which already express boatloads of it. P2A peptides improve both GRN and SMAD, and importantly reduce N-terminal tag to a single aa.
14/ a note of caution is that while background EGFP is minor compared to even the lowest drug induced expression levels, this may need mitigation when using TFs or other strong cell state regulators
November 2, 2025 at 2:30 PM
14/ a note of caution is that while background EGFP is minor compared to even the lowest drug induced expression levels, this may need mitigation when using TFs or other strong cell state regulators
13/ Here's the commonly used H1 ESC background, differentiated into microglia with an embryoid body protocol (we used EB and monolayer)
November 2, 2025 at 2:29 PM
13/ Here's the commonly used H1 ESC background, differentiated into microglia with an embryoid body protocol (we used EB and monolayer)
12/ quantification shows how tunable this system really is! Below the 100nM dose, linear correlations between drug dose and EGFP is basically perfect both before and after differentiation
November 2, 2025 at 2:29 PM
12/ quantification shows how tunable this system really is! Below the 100nM dose, linear correlations between drug dose and EGFP is basically perfect both before and after differentiation
11/ again with AAVS1-targeted EGFP, what we saw was remarkable: first time we've ever seen robust inducibility after long differentiations, this time 4-7 weeks, across hPSC backgrounds and differentiation protocols!
November 2, 2025 at 2:28 PM
11/ again with AAVS1-targeted EGFP, what we saw was remarkable: first time we've ever seen robust inducibility after long differentiations, this time 4-7 weeks, across hPSC backgrounds and differentiation protocols!
9/ we also looked at leakiness, and leaky EGFP varied by system, with Tet-On being pretty tightly controlled (although silenced later on), and degrons showing variability. Note lack of degron tunability in panel C.
November 2, 2025 at 2:27 PM
9/ we also looked at leakiness, and leaky EGFP varied by system, with Tet-On being pretty tightly controlled (although silenced later on), and degrons showing variability. Note lack of degron tunability in panel C.
8/ We next tested degrons, as this have been used successfully for transgene expression specifically in fully differentiated cells. However, their leakiness, tunability, and inducibility hasn't been thoroughly tested.
November 2, 2025 at 2:24 PM
8/ We next tested degrons, as this have been used successfully for transgene expression specifically in fully differentiated cells. However, their leakiness, tunability, and inducibility hasn't been thoroughly tested.
7/ all approaches used EGFP after AAVS1 safe harbor targeting. While these systems worked in hPSCs (albeit with slower kinetics), they still got silenced during differentiation
November 2, 2025 at 2:24 PM
7/ all approaches used EGFP after AAVS1 safe harbor targeting. While these systems worked in hPSCs (albeit with slower kinetics), they still got silenced during differentiation
6/ we then tried to fix this by adding a Tet-Off-TET1 fusion protein, as DNA methylation has been proposed to underlie Tet-On silencing. Here's the idea, with existing systems (top) getting silenced before DOX is added, and new systems (bottom) we hoped would fix it
November 2, 2025 at 2:23 PM
6/ we then tried to fix this by adding a Tet-Off-TET1 fusion protein, as DNA methylation has been proposed to underlie Tet-On silencing. Here's the idea, with existing systems (top) getting silenced before DOX is added, and new systems (bottom) we hoped would fix it
5/ First we showed that Tet-On-based transgenes are silenced early during microglia or macrophage differentiation, regardless of transgene delivery method, transgene components, hPSC background, or differentiation protocol
November 2, 2025 at 2:21 PM
5/ First we showed that Tet-On-based transgenes are silenced early during microglia or macrophage differentiation, regardless of transgene delivery method, transgene components, hPSC background, or differentiation protocol
29/ Finally, we compared these DEGs with those from MS patient microglia from gray matter, white matter, active/inactive lesions, etc. We found a significant overlap, suggesting that iPSC-microglia can recapitulate some MS-relevant pathways when perturbing risk genes at individual loci.
June 22, 2025 at 9:47 PM
29/ Finally, we compared these DEGs with those from MS patient microglia from gray matter, white matter, active/inactive lesions, etc. We found a significant overlap, suggesting that iPSC-microglia can recapitulate some MS-relevant pathways when perturbing risk genes at individual loci.
28/ we also find striking concordance between DEGs across these 5 loci - many genes were shared and directionally concordant. Homeostatic microglia pathways such as GTPase activity were downregulated in cells with MS risk gene perturbation, whereas oxphos and other metabolic pathways were increased.
June 22, 2025 at 9:45 PM
28/ we also find striking concordance between DEGs across these 5 loci - many genes were shared and directionally concordant. Homeostatic microglia pathways such as GTPase activity were downregulated in cells with MS risk gene perturbation, whereas oxphos and other metabolic pathways were increased.
27/ this wasn't just due to altering microglia genes in general, as AD heritability was not nearly as enriched, despite the strong connection of AD SNP heritability with microglia. The effect persisted when excluding the cis target gene and HLA.
June 22, 2025 at 9:44 PM
27/ this wasn't just due to altering microglia genes in general, as AD heritability was not nearly as enriched, despite the strong connection of AD SNP heritability with microglia. The effect persisted when excluding the cis target gene and HLA.
25/ and three other loci: CD86, IRF5, and SLC25A19, the latter of which we show GRB2 as the likely SNP target gene
June 22, 2025 at 9:40 PM
25/ and three other loci: CD86, IRF5, and SLC25A19, the latter of which we show GRB2 as the likely SNP target gene
24/ complement C3, which is regulated by upstream enhancers with MS SNPs, but no effect on TNFSF14 which is actually closer to the GWAS signal......
June 22, 2025 at 9:40 PM
24/ complement C3, which is regulated by upstream enhancers with MS SNPs, but no effect on TNFSF14 which is actually closer to the GWAS signal......
23/ we identified the causal MS risk gene at 5/9 loci, including MERTK, a well know regulatory of phagocytosis...
June 22, 2025 at 9:39 PM
23/ we identified the causal MS risk gene at 5/9 loci, including MERTK, a well know regulatory of phagocytosis...
22/ when we performed the screen, the microglia were very homogeneous, and even lacked markers of highly similar border-associated macrophages like LYVE1. We used MOI of 1.5, sequenced ~120K cells with ~10B read pairs, got ~400 cells/guide and ~2K genes/cell.
June 22, 2025 at 9:38 PM
22/ when we performed the screen, the microglia were very homogeneous, and even lacked markers of highly similar border-associated macrophages like LYVE1. We used MOI of 1.5, sequenced ~120K cells with ~10B read pairs, got ~400 cells/guide and ~2K genes/cell.
21/ Vpx lentiviral system to a Perturb-seq vector for efficient, non-toxic, minimally-activating CRISPRi in #microglia, using dCas9-KRAB with ZIM3 KRAB domain:
June 22, 2025 at 9:38 PM
21/ Vpx lentiviral system to a Perturb-seq vector for efficient, non-toxic, minimally-activating CRISPRi in #microglia, using dCas9-KRAB with ZIM3 KRAB domain:
19/ a really interesting locus if WWOX/MAF, which has both AD and MS GWAS SNPs, as well as MS outside variants, overlapping microglial enhancers spanning 500kb. PLAC-seq in ex vivo microglia and PCHi-C in iPSC-microglia really suggest MAF as the target gene here
June 22, 2025 at 9:35 PM
19/ a really interesting locus if WWOX/MAF, which has both AD and MS GWAS SNPs, as well as MS outside variants, overlapping microglial enhancers spanning 500kb. PLAC-seq in ex vivo microglia and PCHi-C in iPSC-microglia really suggest MAF as the target gene here
18/ so here's what we get when we apply this approach to all 200 non-MHC loci. Outside variant + GWAS SNP enrichments in enhancers suggest that some loci can be resolved to a single cell type of origin, including 17 plausible microglial effect loci.
June 22, 2025 at 9:34 PM
18/ so here's what we get when we apply this approach to all 200 non-MHC loci. Outside variant + GWAS SNP enrichments in enhancers suggest that some loci can be resolved to a single cell type of origin, including 17 plausible microglial effect loci.
16/ the way it works is we 1) look at each GWAS locus, 2) identity putative enhancers that interact with the same target gene as the GWAS region, and 3) determine if there are any SNPs in these "outside" regions that conspire with the GWAS SNPs to further alter risk.
June 22, 2025 at 9:33 PM
16/ the way it works is we 1) look at each GWAS locus, 2) identity putative enhancers that interact with the same target gene as the GWAS region, and 3) determine if there are any SNPs in these "outside" regions that conspire with the GWAS SNPs to further alter risk.
13/ The BIN1 AD risk locus is well characterized, and our iPSC-microglia seem to do a good job recapitulating the epigenomic patterns here.
June 22, 2025 at 9:32 PM
13/ The BIN1 AD risk locus is well characterized, and our iPSC-microglia seem to do a good job recapitulating the epigenomic patterns here.
12/ while we focused on microglia for the rest of the study, here are the s-LDSC results for NPCs, neurons and astrocytes - surprisingly no astrocyte enriched traits!
June 22, 2025 at 9:30 PM
12/ while we focused on microglia for the rest of the study, here are the s-LDSC results for NPCs, neurons and astrocytes - surprisingly no astrocyte enriched traits!