Structural basis for RNA-mediated assembly of type V CRISPR-associated transposons

Schmitz, M. Lienhard; Querques, Irma; Oberli, Seraina; Chanez, Christelle; Jinek, Martin

doi:10.1101/2022.06.17.496590

Cited by 6 publications

(29 citation statements)

References 52 publications

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“…Consistent with TniQ's functional importance in ShCAST transposition (17), TniQ is located at the PAM-distal end of the R-loop close to Cas12k and S15 (Fig. S5) and appears to primarily interact with TnsC and RNA, consistent with the productive recruitment complex (10).…”

Section: Tniq Interactions With Tnsc Are Critical For Cast Transpositionsupporting

confidence: 58%

“…While the orientation and contacts we find in the transpososome assembly are remarkably uniform and well-resolved (Fig. S2) compared to the heterogeneity of other reconstitutions (10), we observe a slight variability in the oligomeric composition of TnsC within transpososome populations. This is not altogether unexpected, since CAST insertion profiles also contain a slight variability (~5 base pairs) in their insertion profiles (2).…”

Section: Discussionmentioning

confidence: 56%

“…The ShCAST recruitment complex is heterogeneous, consisting of both 'productive' and 'nonproductive' complexes, distinguished by the direction of TnsC filaments bound to DNA (10).…”

Section: Dna Substrate Design and Transpososome Reconstitutionmentioning

confidence: 99%

“…100 µL of each pot was mixed to initiate the ATP hydrolysis reaction. For each time point (10,20,30,40,50,60,90 and 120 minutes), 20 µL of the reaction mix was transferred to the 96-well plate (Corning) that contains 5 µL of 0.5 M EDTA to quench further hydrolysis. After taking all the time points, 150 µL of the room-temperature Malachite Green reagent was added to each well, and the plate was shaken for ~5 minutes to develop the color for imaging.…”

Section: Malachite Green Atp Hydrolysis Assaymentioning

confidence: 99%

“…In all Tn7-like systems, a specialized CRISPR effector or DNA binding protein is hypothesized to recruit core transposition proteins sequentially ( 8 ). In CAST elements, TniQ protein associates with the target site via interactions with the CRISPR effector ( 9 ), and in some cases is shown to require a host factor(s) ( 10, 11 ). The AAA+ regulator TnsC serves as a ’matchmaker’ by interacting with both TniQ/TnsD and the transposase (TnsA/B) ( 12, 13 ).…”

Section: Introductionmentioning

confidence: 99%

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Structures of the holo CRISPR RNA-guided transposon integration complex

Park

Tsai

Rizo

et al. 2022

Preprint

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CRISPR-associated transposons (CAST) are programmable mobile elements that insert large DNA cargo by an RNA-guided mechanism. Multiple conserved components act in concert at the target site through formation of an integration complex (transpososome). We reconstituted the type V-K CAST transpososome fromScytonema hofmannii(ShCAST) and determined the structure using cryo-EM. Transpososome architecture ensures orientation-specific association: AAA+ regulator TnsC has defined polarity and length, with dedicated interaction interfaces for other CAST components. Interestingly, transposase (TnsB)-TnsC interactions we observe contribute to stimulating TnsC's ATP hydrolysis activity. TnsC deviates from previously observed helical configurations of TnsC, and target DNA does not track with TnsC protomers. Consequently, TnsC makes new, functionally important protein-DNA interactions throughout the transpososome. Finally, two distinct transpososome populations suggests that associations with the CRISPR effector are flexible. These ShCAST transpososome structures significantly enhances our understanding of CAST transposition systems and suggests avenues for improving CAST transposition for precision genome-editing applications.

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Section: Tniq Interactions With Tnsc Are Critical For Cast Transpositionsupporting

confidence: 58%

Section: Discussionmentioning

confidence: 56%

“…The ShCAST recruitment complex is heterogeneous, consisting of both 'productive' and 'nonproductive' complexes, distinguished by the direction of TnsC filaments bound to DNA (10).…”

Section: Dna Substrate Design and Transpososome Reconstitutionmentioning

confidence: 99%

Section: Malachite Green Atp Hydrolysis Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Structures of the holo CRISPR RNA-guided transposon integration complex

Park

Tsai

Rizo

et al. 2022

Preprint

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Recent Advances in Double-Strand Break-Free Kilobase-Scale Genome Editing Technologies

Tou

Kleinstiver

2022

Biochemistry

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Genome editing approaches have transformed the ability to make user-defined changes to genomes in both ex vivo and in vivo contexts. Despite the abundant development of technologies that permit the installation of nucleotide-level changes, until recently, larger-scale sequence edits via technologies independent of DNA double-strand breaks (DSBs) had remained less explored. Here, we review recent advances toward DSB-free technologies that enable kilobase-scale modifications including insertions, deletions, inversions, replacements, and others. These technologies provide new capabilities for users, while offering hope for the simplification of putative therapeutic strategies by moving away from small mutation-specific edits and toward more generalizable kilobase-scale approaches.

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Transposons and CRISPR: Rewiring Gene Editing

2022

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CRISPR-Cas is driving a gene editing revolution because of its simple reprogramming. However, off-target effects and dependence on the double-strand break repair pathways impose important limitations. Because homology-directed repair acts primarily in actively dividing cells, many of the current gene correction/replacement approaches are restricted to a minority of cell types. Furthermore, current approaches display low efficiency upon insertion of large DNA cargos (e.g., sequences containing multiple gene circuits with tunable functionalities). Recent research has revealed new links between CRISPR-Cas systems and transposons providing new scaffolds that might overcome some of these limitations. Here, we comment on two new transposonassociated RNA-guided mechanisms considering their potential as new gene editing solutions. Initially, we focus on a group of small RNA-guided endonucleases of the IS200/IS605 family of transposons, which likely evolved into class 2 CRISPR effector nucleases (Cas9s and Cas12s). We explore the diversity of these nucleases (named OMEGA, obligate mobile element-guided activity) and analyze their similarities with class 2 gene editors. OMEGA nucleases can perform gene editing in human cells and constitute promising candidates for the design of new compact RNA-guided platforms. Then, we address the co-option of the RNA-guided activity of different CRISPR effector nucleases by a specialized group of Tn7-like transposons to target transposon integration. We describe the various mechanisms used by these RNA-guided transposons for target site selection and integration. Finally, we assess the potential of these new systems to circumvent some of the current gene editing challenges.

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Structural basis for RNA-mediated assembly of type V CRISPR-associated transposons

Cited by 6 publications

References 52 publications

Structures of the holo CRISPR RNA-guided transposon integration complex

Structures of the holo CRISPR RNA-guided transposon integration complex

Recent Advances in Double-Strand Break-Free Kilobase-Scale Genome Editing Technologies

Transposons and CRISPR: Rewiring Gene Editing

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