Extended Data Figure 2. Targeting SNORD115 unsilences paternal UBE3A in human neurons.

a. Alignment of RNA-seq reads from human neural progenitors (top panel) and 8 week differentiated neurons (bottom panel). Colored lines mark polymorphisms not present in the reference genome.

b. Zoom in of the region between dashed lines in a). Arrow denotes SNV (rsID:61734190) used to quantify allele specific expression in Extended Data Figs. 2c,o,p.

c. Allelic expression of UBE3A and UBE3A-ATS before and after differentiation of phNPCs into neurons, calculated as the percentage of stranded RNA-seq reads containing a SNV at chr15:25,371,697 (rsID:61734190).

d. Guide RNA locations in the human SNORD115 cluster.

e. Experimental design for transducing differentiated human neurons with lentiviruses containing SpCas9 and gRNAs.

f. Sorting strategy to isolate differentiated neuron populations from non-neuronal cells. Neurons were labeled and identified using AAV2:hSyn1:EGFP (green), cells lentivirally transduced with Cas9 were identified based on mCherry (red=non-neuronal cells, yellow=neurons). Side scatter counts (SSC), forward scatter counts (FSC).

g-n. Expression of the indicated genes in different cell populations using qPCR and SYBR green, normalized to EIF4A2. Values on Y-axis are presented as ΔCt values (not normalized to a specific sample), so expression values can be directly compared across multiple genes. n=3 for each qPCR reaction.

o. Raw qPCR curves from populations of progenitors and neurons containing the indicated fluorescent markers. Spjw33 negative control (mouse-specific). Presumed maternal (T; red line) and paternal (A; blue line) allele. ΔRn is change in reporter dye intensity per cycle, normalized to passive reference dye. One line per replicate, n=4.

p. Allele specific expression after lentiviral transduction of human neurons with SpCas9 and the indicated gRNAs, relative to neurons from the same cultures that were not transduced with Cas9. Number in parentheses refers to the number of SNORD115 target sites.

q. Expression of the indicated genes from Cas9⁺ neurons transduced with gRNA:hsa#3 versus Cas9⁻ human neurons (dashed line). qPCR, normalized to EIF4A2, n=3. * P<0.01.