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Fig. 1 | Mobile DNA

Fig. 1

From: Unbiased profiling of CRISPR RNA-guided transposition products by long-read sequencing

Fig. 1

Whole-genome SMRT-sequencing resolves simple insertion and cointegrate transposition products. a Left, general mechanism of RNA-guided transposition directed by Cascade (Type I) or Cas12k (Type V). Right, genetic architecture of the E. coli Tn7 transposon, and Tn7-like or Tn5053-like transposons mobilized by Type I-F or Type V-K CRISPR-Cas systems, respectively. Note that Tn5053-like transposons do not encode TnsA. b Roles of TnsB and, when present, TnsA, in DNA excision and integration. Non-replicative cut-and-paste transposition involves both 5′ (TnsA) and 3′ (TnsB) cleavage of the donor DNA, resulting in simple insertion of the mini-transposon (left). Replicative copy-and-paste transposition involves only 3′ cleavage, resulting in a Shapiro intermediate and eventual cointegrate product containing duplicated mini-transposon and embedded plasmid backbone (right). c Representative SMRT-seq CCS reads from the wild-type V. cholerae CRISPR-Tn show hallmarks of simple insertion products (left); SMRT-seq reads from a D90A-TnsA mutant show hallmarks of cointegrates (right). d Population-level quantification of simple insertion and cointegrate transposition products from V. cholerae CRISPR-Tn using either WT TnsA or D90A-TnsA mutant (left), and the WT S. hofmannii CRISPR-Tn (ShCAST) programmed by two distinct guide RNAs (right). Data represent mean ± s.d. for 3 biological replicates; n denotes the total number of CCS reads. e Specificity of RNA-guided transposition determined from SMRT-seq data, for both V. cholerae and S. hofmannii CRISPR-Tn systems. Data are shown as mean for 3 biological replicates; n denotes the total number of CCS reads

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