a, Distribution of NGG and NAG PAM sgRNAs mapped to genomic cleavage position. The vertical lines represent cleavage sites for sgRNAs mapped to plus and minus strands. b, Gap distance between adjacent genomic cleavage position for NAG PAM sgRNAs. c, Library composition by target sequence and PAM restriction. d, Representation of both NGG and NAG sgRNA (1,338 sgRNAs in total) within the plasmid pool by deep sequencing. The median was 718 normalized reads and the 10th and 90th percentiles (indicated by the vertical dotted lines) ranged from 337 to 1,205 normalized reads. e, HbF distribution in HUDEP-2 cells transduced with Cas9 and individual sgRNAs, either non-targeting or targeting BCL11A exon 2. f, HbF enrichment scores of NGG sgRNAs in six biological replicates. g, Sort of library-transduced cells into HbF-high and HbF-low pools. h, Control sgRNA enrichment. Boxes demonstrate 25th, median, and 75th percentiles and whiskers minimum and maximum values. ****P < 0.0001, NS, non-significant. i, NGG sgRNA representation in plasmid pool and cells at conclusion of experiment (left), and in HbF-high and HbF-low pools (right), with dotted lines at x = y and x = 8y. j, Quantile–quantile plots of NGG sgRNA enrichment scores. k, Cellular dropout scores of NGG sgRNAs relative to genomic cleavage position and repetitive elements. Non-targeting sgRNAs pseudo-mapped with 5-bp spacing.