Fig. 1

CRISPR-Cas3 system mediates DNA cleavage in human cells. a Type I-E CRISPR effector is composed of crRNA, Cas3, and a large Cascade complex, which contains Cas5, Cas6, multiple Cas7, Cas8 (Cse1) recognizing the PAM, and two Cas11 (Cse2). b Schematic of the single strand annealing (SSA) assay used to evaluate DNA cleavage and annealing activity. After the transfection of 293T cells with individual Cas, crRNA, and reporter plasmids, dual luciferase activities (Firefly (Fluc) as a reporter and Renilla (Rluc) as the internal control) were sequentially measured (see Supplementary Fig. 2a). c Efficiencies of two plasmid sequences of pre-crRNA, pLRSR, which includes a leader, repeats and a single spacer, and pRSR, which includes repeats and a spacer, both transcribe pre-crRNA, and plasmids of mat-crRNA, pSR (see Supplementary Fig. 3b). Data are presented as mean ± SD. RLU relative light units. *P < 0.05, **P < 0.01, ANOVA with post-hoc Tukey test. d A series of SSA assays lacking specified components of the Cascade-Cas3 effector complex. Lipofection of the pre-crRNA and six Cas (3, 5–8, and 11)-coding plasmids was performed in 293T cells. crRNAnt nontarget crRNA. e Effect of PAM sequences on Cas3-mediated DNA cleavage activity in 293T cells (see Supplementary Table 1). f Effect of a single mismatch (gray) for 32-nt spacer sequences on Cas3-mediated DNA cleavage activity (see Supplementary Table 1). g Effect of Cas3 mutants in the HD nuclease domain (H74A) or in SF2-helicase domain motif 1 (K320N) or motif III (S483/T485A)³¹. h Comparison of DNA cleavage activity between Class 1 E. coli type I-E, S. putrefaciens type I-F, P. furiosus type I-G (Cas3), and Class 2 S.pyogenes type II-A (Cas9) (see Supplementary Table 1 and Supplementary Fig. 4). Source data are in the Source Data file.