Evolutionary and mechanistic diversity of Type I-F CRISPR-associated transposons

Klompe, Sanne E.; Jaber, Nora; Beh, Leslie Y.; Mohabir, Jason; Bernheim, Aude; Sternberg, Samuel H.

doi:10.1016/j.molcel.2021.12.021

Cited by 49 publications

(81 citation statements)

References 68 publications

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“…These observations suggest that, despite the fact that overall DNA-binding mode is likely shared among TnsB proteins, the residues involved in protein-DNA interactions may be different. This is in line with the fact that even with closely related elements (i.e., within the I-F3 CRISPR-Cas elements) ends can diverge sufficiently to allow them to be orthogonal ( Klompe et al, 2022 ). This orthogonality is required for evolving the vast diversity of Tn7-like elements ( Benler et al, 2021 ).…”

Section: Resultssupporting

confidence: 64%

Structural basis of transposon end recognition explains central features of Tn7 transposition systems

Kaczmarska¹,

Czarnocki-Cieciura²,

Górecka-Minakowska³

et al. 2022

Molecular Cell

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Section: Resultssupporting

confidence: 64%

Structural basis of transposon end recognition explains central features of Tn7 transposition systems

Kaczmarska¹,

Czarnocki-Cieciura²,

Górecka-Minakowska³

et al. 2022

Molecular Cell

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“…The DNA-targeting effectors bind to target sites specified by the crRNA guides, recruiting the transposon machinery to catalyze transposon DNA insertion at a fixed distance downstream of the specific target DNA site. As CRISPR-associated transposon (CAST) systems often contain additional defense systems as cargos (Klompe et al, 2022), these elements have been hypothesizes to mediate horizontal gene transfer of host defense systems within bacterial populations by using other transposons and plasmids as shuttle vectors. While crRNAs encoded from the CRISPR arrays guide transposon insertion preferentially into other mobile genetic elements, atypical delocalized crRNAs target integration into host chromosomal sites for transposon homing (Saito et al, 2021) For type V-K systems, RNA-guided transposition relies on the CRISPR effector complex comprising Cas12k, a crRNA and a trans-activating RNA (tracrRNA), and three transposon proteins: the AAA+ ATPase TnsC, the transposase TnsB and the zinc-finger protein TniQ (Strecker et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

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

Schmitz

Querques

Oberli

et al. 2022

Preprint

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CRISPR systems have been co-opted by Tn7-like elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprising RNA-guided Cas12k, TniQ, TnsC and, unexpectedly, the ribosomal protein S15. Complex assembly on target DNA results in complete R-loop formation mediated by critical interactions between TniQ and the trans-activating crRNA, and is coupled with TniQ-dependent nucleation of a TnsC filament. In vivo transposition assays corroborate our structural findings, and biochemical and functional analyses of S15 supports its role as a bona fide component of the type V crRNA-guided transposition machinery. Altogether, our work uncovers key aspects of the mechanisms underpinning RNA-mediated assembly of CRISPR-associated transposons that will guide their development as programmable site- specific gene insertion tools.

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“…A change in regulation may again be the answer, if using the same editor with just a different guide-RNA for a particular disorder could be considered safe without requiring full clinical trials to treat each mutant allele. For safer knock-ins, CRISPR-based transposon systems seem poised to overtake nucleases for this function (Klompe et al, 2022;Pallarès-Masmitjà et al, 2021). Nucleases might be considered a "first-generation" technology for targeted insertion of large sequences such as genes because they only create a double-strand break and leave subsequent steps to the many DNA repair pathways in the cell, which is the source of unexpected events.…”

Section: Editor Challengesmentioning

confidence: 99%

The promise of gene editing: so close and yet so perilously far

Segal

2022

Front. Genome Ed.

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and indels are produced only by the action of the nuclease, have been deregulated in many countries. An exception are those countries within the European Union, where, despite being the third largest producer of genetically engineered crops behind China and the USA, SDN1 crops remain subject to the stringent regulations for genetically modified organisms (GMOs). Such stringent regulations are considered to have a dampening effect on agriculture innovation in the EU, and are perhaps similar to the dampening effect of long regulatory delays on the genetic engineering of livestock animals (Van Eenennaam et al., 2021). Since the first report of genetic engineering in livestock animals in 1985, only a single food animal has been commercialized. This is in part due to the USA Food and Drug Administration and their EU counterparts classifying any intentional altered genomic DNA in animals as an investigational new animal drug (INAD) that is not generally recognized as safe. However, there is a growing realization that the current EU policy towards SDN1 crops needs to be updated (Dima et al., 2022), giving hope to the wider use of these directed editing methods that can dramatically accelerate the production of new varieties compared to traditional breeding techniques.Interestingly, regulations have not hindered innovation in the application of genetic engineering to human health. In fact, this area has been a significant driver of technological advances. Recent publications and scientific meetings, such as the Keystone Symposium on Precision Genome Engineering and the American Society for Gene and Cell Therapy Annual Meeting, highlight the rapid advances in genome editing tools, driven in large part by a sense that new treatments for human disease enabled by these tools are just around the corner. Indeed, by some estimates there are over 100 products using genome editors now in clinical trial (CRISPR Medicine News), led by companies, such as CRISPR Therapeutics, Intellia Therapeutics, Sangamo Therapeutics, Editas Medicine, Precision Biosciences, Caribou Biosciences, Locus Biosciences, and many others. In the academic sector, Phase 1 of the NIH Somatic Cell Genome Editing Consortium (Saha et al., 2021), which had focused primarily on developing new editors and delivery methods, has led to a Phase 2 that is primarily focused on using these tools to

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Evolutionary and mechanistic diversity of Type I-F CRISPR-associated transposons

Cited by 49 publications

References 68 publications

Structural basis of transposon end recognition explains central features of Tn7 transposition systems

Structural basis of transposon end recognition explains central features of Tn7 transposition systems

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

The promise of gene editing: so close and yet so perilously far

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