Skip to main content
. Author manuscript; available in PMC: 2023 Aug 11.
Published in final edited form as: Nat Biotechnol. 2023 Jan 16:10.1038/s41587-022-01613-7. doi: 10.1038/s41587-022-01613-7

Fig. 1. High-throughput screen for determinants of prime-editing efficiency.

Fig. 1

(a) pegRNA visualization with different pegRNA domains. Example edit is depicted in red (A:T) at position 5 of the RTT domain, leading to a G to T base change in the target DNA. (b) Self-targeting construct with the promoter (hU6) and different pegRNA domains, target sequence, and primer location for NGS-PCR (Fw and Rv primer). (c) Visualization of the workflow of the self-targeting screen in HEK293T cells. (d) Effect of the maximum number of consecutive 'T' in spacer sequence or pegRNA extension on editing efficiency. (e-f) Comparison of edits with different insertion- and deletion-length on editing efficiency. (g) Effect of replacement bases in single base replacement edits on editing efficiency. (h) Heatmap visualizing editing efficiency of pegRNAs (single base replacements) with different RTT overhang lengths (3, 7, 10, 15 bp) and edit positions (1-15); PAM position highlighted with black rectangle. (i) Heatmap visualizing unintended editing rate of pegRNAs (single base replacements) with different RTT overhang lengths (3, 7, 10, 15 bp) and edit positions (1-15); PAM position highlighted with black rectangle. The number of analyzed pegRNA-target combinations are as follows: (d) n = 12,005, 42,611, 24,574, 9,223, 2,918, 1,092. (e) n = 21,882, 2,934, 1,103, 2,501. (f) n = 5,457, 459, 115, 52. (g) n = 4,482, 5,892, 6,576, 3,622, 5,252, 3,768, 4,868, 4,644, 4,864, 5,767, 4,832, 3,353. (h-i) n = 57,920. Boxplots in (d-g) represent the 25th, 50th, and 75th percentiles. Whiskers indicate 5 and 95 percentiles.