Skip to main content
. 2022 Jun 27;13:3685. doi: 10.1038/s41467-022-31322-3

Fig. 1. De novo L1-ORFeus insertions at CRISPR/Cas9 target sites in a reverse transcriptase (RT) dependent manner.

Fig. 1

a De novo L1-ORFeus retrotransposition reporter. The L1-ORFeus expression plasmid contains an eGFP cassette in the L1 3′UTR which is in the opposite orientation of the L1 and is interrupted by an intron. Cells express eGFP only after the L1-ORFeus transcript undergoes splicing and intron removal, reverse transcription, and integration into chromosomal DNA. b Schematic of the experimental strategy employed to examine de novo L1-ORFeus retrotransposition events at the MYC CRISPR/Cas9 target site in HEK293T cells. c Pie charts show the relative abundance of three main types of obtained by amplicon sequencing at the MYC locus targeted by MYC CRISPR/Cas9 in HEK293T cells expressing L1-ORFeus, L1-ENm, L1-RTm, or GFP control. d Numbers of L1-ORFeus insertions obtained by amplicon sequencing at the MYC locus targeted by MYC CRISPR/Cas9 (d, left) or at the RAG1 locus targeted by RAG1 CRISPR/Cas9 (d, right) in HEK293T cells expressing L1-ORFeus, L1-ENm, L1-RTm, or GFP control. Number of L1-ORFeus insertions was pooled from 4 independent experiments with similar results. e Numbers and fragment lengths of L1-ORFeus insertions obtained by amplicon sequencing at the MYC locus targeted by MYC CRISPR/Cas9 in HEK293T cells expressing L1-ORFeus, L1-ENm, L1-RTm, or GFP control. The X-axis indicates the nucleotide position of the full-length L1 (ORFeus) reporter cassette. Top: each histogram represents the number of L1 insertions mapping to a 100 bp interval of the L1 (ORFeus) sequence represented on the X-axis. In orange are insertions oriented from 5′ to 3′ end of the L1 (ORFeus) sequence (+), in blue are insertions oriented from 3′ to 5′ (−); bottom: Length of the L1 insertions mapping to the L1 (ORFeus) sequence represented on the X-axis. Each dot indicates the center of the insertion fragment oriented either 5′ to 3′ (+) or 3′ to 5′ (−). Each bar represents the projection of the dot to indicate the nucleotide position in the full-length L1 (ORFeus) reporter cassette. Example L1-ORFeus insertions bridging the intron of GFP in cells expressing L1-ORFeus are shown in Supplementary Sequence 6.