Sequestration of LINE‐1 in cytosolic aggregates by MOV10 restricts retrotransposition

A

Schematic depicting full‐length L1 element and summary of CRISPRa. To generate L1^UP cells, mESCs were co‐transfected with plasmid encoding catalytically dead Cas9 protein (dCas9) fused to VP160 and sgRNAs that (1) targeted the fusion protein to the 5'UTR sequence of Tf L1 family allowing (2) recruitment of transcription factors and (3) transcription machinery to (4) upregulate L1 transcription.

B

Sequence alignment of 5'UTR sequences of murine L1 Tf, Gf and A subfamilies. The two sgRNA sequences used to upregulate L1 expression are indicated in blue and green, with protospacer adjacent motifs (PAM) in bold.

C

RT–qPCR analysis to quantitate Dicer, and L1 RNA expression levels in the depicted cell lines. Rrm2 was utilized for normalization, and graphs depict fold change in transcript levels compared to WT which was set to one (n = 3 technical replicates).

D

Representative northern blot analysis probed for L1 RNA to assess L1 transcript length and expression levels in the engineered L1^UP Cl1, Cl2 as compared with Ctrl cells. Arrow points to full‐length L1 transcript. Ethidium bromide staining of 28S RNA was used to confirm equal loading (n = 3 technical replicates).

E

Differential gene expression from RNA‐seq analysis of Dicer_KO vs. WT mESCs plotted using previously published data (Bodak et al, ²⁰¹⁷).

F

RT–qPCR analysis to confirm upregulation of Mov10 in Dicer_KO cells. Rrm2 was utilized for normalization, and graphs depict fold change in transcript levels in the indicated cell lines as compared to WT which was set to one (n = 3 technical replicates).

Figure EV2. Generation of mESCs upregulating L1 expression using CRISPRa.