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. 2022 Nov 17;50(20):11670–11681. doi: 10.1093/nar/gkac985

Figure 2.

Figure 2.

Reconstitution and characterization of RNA-guided DNA integration using a diverse set of Type I-F3 CRISPR-associated transposons. (A) Diagram of the reconstitution of each CAST into a two-plasmid system. A pEffector plasmid contains a CRISPR array, tnsABC, tniQ, and cas876 from the associated transposon. A pDonor plasmid contains cargo DNA flanked by the predicted right and left ends (R and L) of the associated transposon. (B) A lacZ-targeting spacer present within the pEffector CRISPR array drives RNA-guided DNA integration into lacZ. The integration event results in a transposon integrated in either the right-to-left (RL) or left-to-right (LR) orientation. Two primer pairs (ab, bc) were used to quantify integration events by qPCR. (C) RNA-guided DNA integration efficiency was measured by qPCR for eleven CASTs at two different temperatures, 30°C and 25°C, at a single lacZ target site with a 5′-TCC-3′ PAM. (D) Orientation bias data related to (C). (E) Integration distance data, related to the target site from (C), is shown. Integration distance is the number of base pairs between the end of the target site and the start of the integrated transposon. (F) Integration efficiency involving transposons approximately 1 kb and 10 kb in size. Coverage is shown from Nanopore sequencing of genomic DNA derived from an isolate containing the integrated 10 kb transposon. The expanded genomic context is illustrated below. For parts C, D and F, shown are the mean, SD, and individual data points from n = 3 biological replicates.