Dual modes of CRISPR-associated transposon homing

Saito, Makoto; Ladha, Alim; Strecker, Jonathan; Faure, Guilhem; Neumann, Edwin N.; Altae-Tran, Han; Macrae, Rhiannon K.; Zhang, Feng

doi:10.1016/j.cell.2021.03.006

Cited by 101 publications

(180 citation statements)

References 36 publications

(71 reference statements)

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“…22 The V-K system exhibits a high off-target rate, 22,29 an undesirable propensity to cointegrate its donor plasmid 30,31 and relatively low efficiency of multiple integration. 20 The I-B1 system from Anabaena variabili was not observed to have any cointegrate insertions, 32 which indicates its potential for applications of orthogonality with the I-F3 system. However, its integration efficiency is unsatisfactory for engineering purposes.…”

Section: Discussionmentioning

confidence: 98%

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Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Zhang

Yang

et al. 2021

The CRISPR Journal

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Directed evolution and targeted genome editing have been deployed to create genetic variants with usefully altered phenotypes. However, these methods are limited to high-throughput screening methods or serial manipulation of single genes. In this study, we implemented multicopy chromosomal integration using CRISPRassociated transposases (MUCICAT) to simultaneously target up to 11 sites on the Escherichia coli chromosome for multiplex gene interruption and/or insertion, generating combinatorial genomic diversity. The MUCICAT system was improved by replacing the isopropyl-beta-D-thiogalactoside (IPTG)-dependent promoter to decouple gene editing and product synthesis and truncating the right end to reduce the leakage expression of cargo. We applied MUCICAT to engineer and optimize the N-acetylglucosamine (GlcNAc) biosynthesis pathway in E. coli to overproduce the industrially important GlcNAc in only 8 days. Two rounds of transformation, the first round for disruption of two degradation pathways related gene clusters and the second round for multiplex integration of the GlcNAc gene cassette, would generate a library with 1-11 copies of the GlcNAc cassette. We isolated a best variant with five copies of GlcNAc cassettes, producing 11.59 g/L GlcNAc, which was more than sixfold than that of the strain containing the pET-GNAc plasmid. Our multiplex approach MUCICAT has potential to become a powerful tool of cell programing and can be widely applied in many fields such as synthetic biology.

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Section: Discussionmentioning

confidence: 98%

“…However, its integration efficiency is unsatisfactory for engineering purposes. 32 Therefore, a set of orthogonal systems that meets the requirements of strain engineering still needs to be developed.…”

Section: Discussionmentioning

confidence: 99%

Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Zhang

Yang

et al. 2021

The CRISPR Journal

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“…Furthermore, the R-loop structure is compatible with the reported minimal requirements for guide RNAtarget complementarity in ShCAST 6 . The structure of TnsC filaments shows that ATP-dependent TnsC polymerization occurs on remodeled, underwound DNA, hinting that DNA unwinding and R-loop formation by Cas12k may help nucleate TnsC filament formation, although R-loop formation alone is not sufficient for ShCAST activity 7 .…”

Section: Discussionmentioning

confidence: 54%

“…Prokaryotes have evolved genome defense mechanisms to restrict parasitic transposition, including adaptive immunity provided by CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) systems that rely on Cas proteins and CRISPR RNA (crRNA) guides to target invasive genetic elements for nucleolytic degradation 1,10 . In contrast to the defensive role of canonical CRISPR-Cas systems, several nucleasedeficient type I-F, I-B and V-K systems have been instead co-opted by a distinct group of Tn7-like transposons to direct RNA-guided transposon DNA insertion into specific target sites [2][3][4][5][6][7] . In these systems, DNA targeting relies on transposon-encoded Cas-RNPs, involving a Cas3-less multisubunit effector complex (termed Cascade) in type I systems 3 or a single catalytically inactive Cas12k protein in type V-K systems 7 .…”

Section: Introductionmentioning

confidence: 99%

“…

Although the canonical function of CRISPR-Cas systems is to provide adaptive immunity against mobile genetic elements 1 , type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion [2][3][4][5][6][7] . Type V-K CRISPR-associated transposons rely on the activities of the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ 7 .

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mentioning

confidence: 99%

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Molecular mechanism of target site selection and remodeling by type V CRISPR-associated transposons

Querques

Schmitz

Oberli

et al. 2021

Preprint

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Although the canonical function of CRISPR-Cas systems is to provide adaptive immunity against mobile genetic elements, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion. Type V-K CRISPR-associated transposons rely on the activities of the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ. However, the molecular and structural details of RNA-directed DNA transposition have remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. The assembly of the TnsC helical filament is ATP-dependent and accompanied by structural remodeling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments further show that TniQ restricts TnsC polymerization, while the TnsB transposase interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest a mechanistic model whereby RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodeling, generating a recruitment platform for TnsB to catalyze site-specific transposon insertion. The present work advances our mechanistic understanding of the cross-talk between CRISPR effectors and the transposition machinery and will inform design efforts to harness CRISPR-associated transposons as programmable site-specific gene insertion tools for genome engineering applications.

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DNA on the move: mechanisms, functions and applications of transposable elements

Schmitz,

Querques

2023

FEBS Open Bio

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Transposons are mobile genetic elements that have invaded all domains of life by moving between and within their host genomes. Due to their mobility (or transposition), transposons facilitate horizontal gene transfer in bacteria and foster the evolution of new molecular functions in prokaryotes and eukaryotes. As transposition can lead to detrimental genomic rearrangements, organisms have evolved a multitude of molecular strategies to control transposons, including genome defense mechanisms provided by CRISPR‐Cas systems. Apart from their biological impacts on genomes, DNA transposons have been leveraged as efficient gene insertion vectors in basic research, transgenesis and gene therapy. However, the close to random insertion profile of transposon‐based tools limits their programmability and safety. Despite recent advances brought by the development of CRISPR‐associated genome editing nucleases, a strategy for efficient insertion of large, multi‐kilobase transgenes at user‐defined genomic sites is currently challenging. The discovery and experimental characterization of bacterial CRISPR‐associated transposons (CASTs) led to the attractive hypothesis that these systems could be repurposed as programmable, site‐specific gene integration technologies. Here, we provide a broad overview of the molecular mechanisms underpinning DNA transposition and of its biological and technological impact. The second focus of the article is to describe recent mechanistic and functional analyses of CAST transposition. Finally, current challenges and desired future advances of CAST‐based genome engineering applications are briefly discussed.

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Dual modes of CRISPR-associated transposon homing

Cited by 101 publications

References 36 publications

Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Programming Cells by Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Molecular mechanism of target site selection and remodeling by type V CRISPR-associated transposons

DNA on the move: mechanisms, functions and applications of transposable elements

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