CRISPRで遺伝子の “ヒッチハイカー”を誘導することに成功(Genetic ‘Hitchhikers’ Can Be Directed Using CRISPR)


2022-11-21 ノースカロライナ州立大学(NCState)



多様なタイプI-F CRISPR関連トランスポゾンの機能解析 Functional characterization of diverse type I-F CRISPR-associated transposons

Avery Roberts, Matthew A Nethery, Rodolphe Barrangou
Nucleic Acids Research  Published:17 November 2022

Form and function of Type I-F3 CRISPR-associated transposons. (A) Tn7479 is shown as a representative Type I-F3 CAST. Genes essential for transposition, cargo genes not essential for transposition, the att site (rsmJ), and transposon ends (R and L) are labeled. The atypical repeat of the CRISPR array is colored light grey. (B) A detailed view of the transposon ends and CRISPR array of Tn7479 in (A). The eight putative TnsB binding site sequences were used to generate a consensus WebLogo (top). 5-bp target site duplication events (black) indicative of Tn7-like transposition events and the terminal inverted repeats (green) that define the ends of the transposon are shown alongside the TnsB binding sites (purple arrows) within the transposon ends. The CRISPR array contains a self-targeting spacer that is complementary to a region of the att site (rsmJ) and flanked by an atypical repeat (light grey diamond). The rsmJ target site and self-targeting spacer possess mismatches colored in red. (C) A mechanistic overview of Type I-F3 CRISPR RNA-guided DNA transposition. The TniQ-Cascade complex is guided to the target site complementary to the spacer sequence of the bound crRNA. TnsB proteins bind to sites present in the transposon ends and transposition, regulated by TnsC activity between the TniQ-Cascade complex and the heteromeric transposase TnsAB, results in integration of the transposon ∼50 bp downstream from the end of the target site. (D) A phylogenetic tree of representative TnsB clades is shown with Tn7 as an outgroup and eleven diverse Type I-F3 CASTs selected for characterization. (E) The cargo genes of the eleven Type I-F3 CASTs selected for characterization were used to generate a heatmap displaying the gene counts for each Clusters of Orthologous Genes (COG) category.


CRISPR-Cas systems generally provide adaptive immunity in prokaryotes through RNA-guided degradation of foreign genetic elements like bacteriophages and plasmids. Recently, however, transposon-encoded and nuclease-deficient CRISPR-Cas systems were characterized and shown to be co-opted by Tn7-like transposons for CRISPR RNA-guided DNA transposition. As a genome engineering tool, these CRISPR-Cas systems and their associated transposon proteins can be deployed for programmable, site-specific integration of sizable cargo DNA, circumventing the need for DNA cleavage and homology-directed repair involving endogenous repair machinery. Here, we selected a diverse set of type I-F3 CRISPR-associated transposon systems derived from Gammaproteobacteria, predicted all components essential for transposition activity, and deployed them for functionality testing within Escherichia coli. Our results demonstrate that these systems possess a significant range of integration efficiencies with regards to temperature, transposon size, and flexible PAM requirements. Additionally, our findings support the categorization of these systems into functional compatibility groups for efficient and orthogonal RNA-guided DNA integration. This work expands the CRISPR-based toolbox with new CRISPR RNA-guided DNA integrases that can be applied to complex and extensive genome engineering efforts.