Extended Data Fig. 3: Trans-cleavage of non-specific ssDNA directed by crRNA and tracrRNA.
From: Trans-nuclease activity of Cas9 activated by DNA or RNA target binding
a, Comparison of sgRNA and crRNA-tracrRNA guided FnCas9 trans-cleavage on M13 phage ssDNA substrate using a target ssDNA activator. There is no sequence homology between the M13 ssDNA and the target ssDNA. b, crRNA-tracrRNA guided SpyCas9 trans-cleavage assays using 5′-Cy5 labeled non-specific dsDNA-1 substrate with random sequence. c, crRNA-tracrRNA guided SpyCas9 trans-cleavage assays using 5′-Cy5 labeled non-specific ssDNA-2 (left) and 3′-Cy5 labeled non-specific ssDNA-3 (right) substrates with random sequences. Assays in Extended Data Fig. 3a–c were repeated three times independently with similar results. d, Schematic representation of an assay to explore the effect of the trans-cleavage activity of Cas9 on bacterial immunity. e, SpyCas9 can target and delete the pTarget plasmid in Escherichia coli. The pCas9 and pTarget plasmids contain the Chl-resistant gene and Amp-resistant gene, respectively. The cells were serially diluted and dropped onto plates containing indicated antibiotics. Control: The guide sequence of crRNA is not complementary to the target sequence (pTarget). Targeting: The guide sequence of crRNA is complementary to the target sequence (pTarget). f, The trans-cleavage activity of SpyCas9 does not prevent plague formation. Ten-fold serial dilutions of M13 phage were dropped onto bacterial lawns. g, The titers of M13 phage after infecting Escherichia coli. PFU values are shown as mean ± s.d. (n = 3 independent experiments). Statistical analysis was by two-tailed t test.
