Structural basis for the assembly of the type V CRISPR-associated transposon complex

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

doi:10.1016/j.cell.2022.11.009

Cited by 43 publications

(48 citation statements)

References 69 publications

(138 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is striking that, although Type I-F CASTs rely on a multitude of transposon-encoded genes, diverse DNA sequence determinants, and potential additional host-encoded factors, heterologous assays in E. coli with twenty CASTs from a range of gammaproteobacteria revealed active transposition for all (18). How and why mobile genetic elements would evolve dependencies on host-specific factors are questions that encourage further research into the regulation of transposition and search for additional accessory factors (63), especially in native host organisms.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Transposon mutagenesis libraries reveal novel molecular requirements during CRISPR RNA-guided DNA integration

Walker

Klompe

Zhang

et al. 2023

Preprint

View full text Add to dashboard Cite

CRISPR-associated transposons (CASTs) direct DNA integration downstream of target sites using the RNA-guided DNA binding activity of nuclease-deficient CRISPR-Cas systems. Transposition relies on several key protein-protein and protein-DNA interactions, but little is known about the explicit sequence requirements governing efficient transposon DNA integration activity. Here, we exploit pooled library screening and high-throughput sequencing to reveal novel sequence determinants during transposition by the Type I-F Vibrio cholerae CAST system. On the donor DNA, large mutagenic libraries identified core binding sites recognized by the TnsB transposase, as well as an additional conserved region that encoded a consensus binding site for integration host factor (IHF). Remarkably, we found that VchCAST requires IHF for efficient transposition, thus revealing a novel cellular factor involved in CRISPR-associated transpososome assembly. On the target DNA, we uncovered preferred sequence motifs at the integration site that explained previously observed heterogeneity with single-base pair resolution. Finally, we exploited our library data to design modified transposon variants that enable in-frame protein tagging. Collectively, our results provide new clues about the assembly and architecture of the paired-end complex formed between TnsB and the transposon DNA, and inform the design of custom payload sequences for genome engineering applications of CAST systems.

show abstract

Section: Discussionmentioning

confidence: 99%

“…How and why mobile genetic elements would evolve dependencies on host-specific factors are questions that encourage further research into the regulation of transposition and search for additional accessory factors (63), especially in native host organisms.…”

Section: Discussionmentioning

confidence: 99%

Transposon mutagenesis libraries reveal novel molecular requirements during CRISPR RNA-guided DNA integration

Walker

Klompe

Zhang

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Several classic mobile elements, including Tn7 and bacteriophage Mu, also depend on ATP-dependent factors to select the appropriate insertion sites 70 , 72 – 74 . Recently, the structures of a recruitment complex and a holo-transposome from a Tn7-like element have been reported 75 , 76 . Although these studies help to define how the AAA + factor selects the insertion site, the structure of the TnsB tetramer remains largely unchanged and, therefore, how these ‘molecular matchmakers’ precisely regulate the transposase activity to promote DNA transposition remains unclear.…”

Section: Discussionmentioning

confidence: 99%

IS21 family transposase cleaved donor complex traps two right-handed superhelical crossings

et al. 2023

View full text Add to dashboard Cite

Transposases are ubiquitous enzymes that catalyze DNA rearrangement events with broad impacts on gene expression, genome evolution, and the spread of drug-resistance in bacteria. Here, we use biochemical and structural approaches to define the molecular determinants by which IstA, a transposase present in the widespread IS21 family of mobile elements, catalyzes efficient DNA transposition. Solution studies show that IstA engages the transposon terminal sequences to form a high-molecular weight complex and promote DNA integration. A 3.4 Å resolution structure of the transposase bound to transposon ends corroborates our biochemical findings and reveals that IstA self-assembles into a highly intertwined tetramer that synapses two supercoiled terminal inverted repeats. The three-dimensional organization of the IstA•DNA cleaved donor complex reveals remarkable similarities with retroviral integrases and classic transposase systems, such as Tn7 and bacteriophage Mu, and provides insights into IS21 transposition.

show abstract

“…However, we point interested readers to recent studies that describe both mechanistic and technological advances in the study and application of Type V-K CAST systems 45,47,48 .…”

Section: Mechanism and Development Of Type I-f Castsmentioning

confidence: 99%

“…Due to key advantages we previously reported for Type I-F CAST systems 32 , we prioritized subsequent technology development on these systems and focus the remainder of this protocol on providing technical details and guidelines for their use in bacteria. However, we point interested readers to recent studies that describe both mechanistic and technological advances in the study and application of Type V-K CAST systems 45,47,48 .…”

Section: Introductionmentioning

confidence: 99%

Bacterial genome engineering using CRISPR RNA-guided transposases

Gelsinger

Klompe

et al. 2023

Preprint

View full text Add to dashboard Cite

CRISPR-associated transposons (CASTs) have the potential to transform the technology landscape for kilobase-scale genome engineering, by virtue of their ability to integrate large genetic payloads with high accuracy, easy programmability, and no requirement for homologous recombination machinery. These transposons encode efficient, CRISPR RNA-guided transposases that execute genomic insertions in E. coli at efficiencies approaching ~100%, generate multiplexed edits when programmed with multiple guides, and function robustly in diverse Gram-negative bacterial species. Here we present a detailed protocol for engineering bacterial genomes using CAST systems, including guidelines on the available homologs and vectors, customization of guide RNAs and DNA payloads, selection of common delivery methods, and genotypic analysis of integration events. We further describe a computational crRNA design algorithm to avoid potential off-targets and CRISPR array cloning pipeline for DNA insertion multiplexing. Starting from available plasmid constructs, the isolation of clonal strains containing a novel genomic integration event-of-interest can be achieved in 1 week using standard molecular biology techniques.

show abstract

Structural basis for the assembly of the type V CRISPR-associated transposon complex

Cited by 43 publications

References 69 publications

Transposon mutagenesis libraries reveal novel molecular requirements during CRISPR RNA-guided DNA integration

Transposon mutagenesis libraries reveal novel molecular requirements during CRISPR RNA-guided DNA integration

IS21 family transposase cleaved donor complex traps two right-handed superhelical crossings

Bacterial genome engineering using CRISPR RNA-guided transposases

Contact Info

Product

Resources

About