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a, The EvolvR system consists of a CRISPR-guided nickase that nicks the target locus and a fused DNA polymerase that performs error-prone nick translation. b, High-throughput sequencing shows that fusing nCas9 to PolI3M resulted in substitutions across an approximately 17-nucleotide window 3′ from the nick. Expressing nCas9–PolI3M with an off-target guide did not show substitutions at a frequency above our detection threshold (dotted line, see Methods ‘High-throughput sequencing data analysis’), whereas an unfused nCas9 and PolI3M yielded only one substitution and at low-frequency. c, Distribution of the substituted nucleotides; all four nucleotides were substituted by nCas9–PolI3M. d, Schematic of the fluctuation analysis workflow used to sensitively quantify targeted and global mutation rates. e, The global and targeted mutation rates of wild-type (WT) E. coli, nCas9–PolI3M, unfused nCas9 and PolI3M, PolI3M alone, and nCas9 alone were determined by fluctuation analysis. For all figures, ‘on target’ mutation rates were determined by expressing a gRNA that nicks 11 nucleotides 5′ of the nonsense mutation unless labelled otherwise, whereas the ‘off target’ mutation rates were determined by expressing a gRNA targeting dbpA, a fitness-neutral RNA helicase gene in the E. coli genome. Data are the mean of ten biologically independent samples and the error bars indicate 95% confidence intervals. Mutation rates throughout are mutations per nucleotide per generation.
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