The majority of genetic variants affecting complex traits map to regulatory regions of genes, and typically lie in credible intervals of 100 or more SNPs. Fine mapping of the causal variant(s) at a locus depends on assays that are able to discriminate the effects of polymorphisms or mutations on gene expression. Here, we evaluated a moderate-throughput CRISPR-Cas9 mutagenesis approach, based on replicated measurement of transcript abundance in single-cell clones, by deleting candidate regulatory SNPs, affecting four genes known to be affected by large-effect expression Quantitative Trait Loci (eQTL) in leukocytes, and using Fluidigm qRT-PCR to monitor gene expression in HL60 pro-myeloid human cells. We concluded that there were multiple constraints that rendered the approach generally infeasible for fine mapping. These included the non-targetability of many regulatory SNPs, clonal variability of single-cell derivatives, and expense. Power calculations based on the measured variance attributable to major sources of experimental error indicated that typical eQTL explaining 10% of the variation in expression of a gene would usually require at least eight biological replicates of each clone. Scanning across credible intervals with this approach is not recommended.

The regenerative ability of planarians depends largely on its complex signaling pathways. The Wnt pathway regulates the anterior/posterior (A/P) polarity formation after regeneration, while the MAPK pathway plays a role in anterior regeneration. This experiment uses various drugs to disrupt the aforementioned pathways. Imatinib targets the receptor tyrosine kinases (RTKs), a common type of surface receptors that play a role in the Wnt pathway. PZQ is expected to affect the Wnt noncanonical calcium pathway. EHT 1864 inhibits Rac1, a GTPase involved in the noncanonical PCP pathway. Finally, U0126 disrupts the MAPK pathway and blastemic cell differentiation. After drug treatment, abnormal planarian regeneration is expected. The drug assays demonstrated that while both Imatinib and PZQ have no effect on planarian regeneration, EHT 1864 under high concentration has a potent effect on the viability of planarians during regeneration. Furthermore, U0126 caused cyclopia, a condition in which organisms only develop one eye instead of the normal number, in planarians under high concentrations. These observations suggest that the RTKs play a limited role in planarian regeneration, Rac1 plays a greater role than just A/P determination during regeneration, and that U0126 affects eye and head regeneration. Our assays with PZQ also show that different species of planarians might have different noncanonical calcium pathways.L'abilité regénérative des planaires dépend largement sur la complexité des ses voies de signalisation. La voie des Wnt contrôle la formation de la polarité des potentiels d'action après la regénération, alors que la voie de la MAPK joue un rôle dans la regénération. L'imatinib cible les RTK, eux-mêmes jouant un rôle dans la voie des Wnt. Praziquantel est attendu d'affecter la voie de calcium non canonique des Wnt. L'EHT 1864 inhibe la Rac1, un GTPase impliqué dans la voie non canonique du PCP. Finalement, U0126 perturbe la voie de la MAPK, l'activité de laquelle induit la différentiation des cellules souches blastémiques. Après traitement avec de la drogue, de la regénération anormale des planaires est attendue. Les essais des drogues ont démontré que, bien que Imatinib et PZQ n'ont pas d'effets sur la regénération des planaires, l'EHT 1864 en haute concentration a un effet potent sur la viabilité des planaires durant la regénération. De plus, l'U0126 a causé la cyclopie chez les planaires en haute concentration. Ces observations suggère que les RTK jouent un rôle limité dans la regénération de planaires, la Rac1 joue un rôle plus important que simplement déterminer des potentiels d'action durant la regénération et l'U0126 affecte les regénérations des yeux et de la tête. Nos découvertes indiquent aussi des incohérences avec une étude par un autre groupe au sujet des effets du PZQ sur la formation polaire des planaires.