(A) Distribution of the position of the 5′ end of the anchor along cassette-associated expressed canonical gRNA genes. Position 1 is the 5′ end of the 5 nt initiation sequence (i.e., anchor completely overlaps initiation sequence). Anchors of most gRNA genes overlap the initiation sequence by 1 nt (position 5) or are separated from the initiation sequence by 1 nt (position 7). (B) For genes whose anchors do not completely overlap the initiation sequence (position ≥ 2), mean anchor length reduces by 0.6 nt (95%CI 5.4–6.9 nt) for each nucleotide separating the anchor from the start of the gene (t = 16.0, P < 0.001). (C) Also for these genes, the 3′ end position of an anchor is weakly correlated with its 5′ start position (R2 = 0.1). Guide RNA genes whose anchors completely overlap the initiation sequence (position 1) do not follow this pattern. Circles: data for individual genes with jitter added to separate circles; black lines: linear regression lines; dotted lines in C: 70% of genes have 3′ anchor positions between 15 and 19 nt from the start of the gene.
