Fig. 1: Genome editing activity of CRISPR type I-D detected by luc reporter assay.

a The CRISPR type I-D (TiD) structure. Upper; subunit organization of TiD and schematic of gRNA (black) of TiD. Middle; schematic of gRNA (blue) of TiD and target DNA (black). The PAM of the target is shown in red. b PAM identification by the E. coli negative selection screening using ccdB expression system. PAM library was inserted in front of the target sequence of ccdB promoter. PAM frequency was determined using the survived E. colli cells. Data are means ± S.E. of independent experiments (n = 3). c Scheme of the luciferase reporter assay used to detect genome editing in human HEK293T cells. The Cas expression vectors and a LUC reporter vector, in which the target sequence was introduced, were transfected into HEK293T cells, and endonuclease cleavage was detected by luminescence. d Luc reporter assay showed both Cas3d and Cas10d were required for the TiD activity. Black bar; non-target gRNA in the luc reporter assay. gRNAs were target to the human AAVS locus listed in Supplementary Table 1. Data are means ± S.E. of independent experiments (n = 4). *P < 0.05 and **P < 0.01 are determined by Student’s t tests. e, Effect of the gRNA target sequence length in the TiD activity. Data are means ± S.E. of independent experiments (n = 4). *P < 0.05, **P < 0.01, and ***P < 0.005 are determined by Student’s t tests. f Luc reporter assay to determine targets for the SlIAA9 gene. gRNAs were target to the tomato IAA9 gene (SlIAA9) listed in Supplementary Table 1. Data are means ± S.E. of independent experiments (n = 4) and *P < 0.05, **P < 0.01, and ***P < 0.005 are determined by Student’s t tests.