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1.
Nat Protoc ; 19(3): 752-790, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38216671

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated transposases 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 Escherichia coli at efficiencies approaching ~100%. Moreover, they 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 CRISPR-associated transposase (CAST) systems, including guidelines on the available vectors, customization of guide RNAs and DNA payloads, selection of common delivery methods, and genotypic analysis of integration events. We further describe a computational CRISPR RNA design algorithm to avoid potential off-targets, and a CRISPR array cloning pipeline for performing multiplexed DNA insertions. The method presented here allows the isolation of clonal strains containing a novel genomic integration event of interest within 1-2 weeks using available plasmid constructs and standard molecular biology techniques.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Transposases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Transposases/genética , RNA Guia de Sistemas CRISPR-Cas , Genoma Bacteriano , DNA , Escherichia coli/genética , Sistemas CRISPR-Cas/genética , Engenharia Genética/métodos , Edição de Genes
2.
Nat Biotechnol ; 42(1): 87-98, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36991112

RESUMO

Conventional genome engineering with CRISPR-Cas9 creates double-strand breaks (DSBs) that lead to undesirable byproducts and reduce product purity. Here we report an approach for programmable integration of large DNA sequences in human cells that avoids the generation of DSBs by using Type I-F CRISPR-associated transposases (CASTs). We optimized DNA targeting by the QCascade complex through protein design and developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase TnsC to genomic sites targeted by QCascade. After initial detection of plasmid-based integration, we screened 15 additional CAST systems from a wide range of bacterial hosts, identified a homolog from Pseudoalteromonas that exhibits improved activity and further increased integration efficiencies. Finally, we discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, likely by promoting active disassembly of the post-integration CAST complex, akin to its known role in Mu transposition. Our work highlights the ability to reconstitute complex, multi-component machineries in human cells and establishes a strong foundation to exploit CRISPR-associated transposases for eukaryotic genome engineering.


Assuntos
Sistemas CRISPR-Cas , Transposases , Humanos , Sistemas CRISPR-Cas/genética , Transposases/genética , Plasmídeos , DNA , Genoma , Edição de Genes
3.
Nucleic Acids Res ; 51(9): 4519-4535, 2023 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-37078593

RESUMO

CRISPR-associated transposases (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 (VchCAST). On the donor DNA, large transposon end libraries revealed binding site nucleotide preferences for 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 with CAST systems.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Elementos de DNA Transponíveis , RNA , Sítios de Ligação/genética , Elementos de DNA Transponíveis/genética , Fatores Hospedeiros de Integração/genética , Transposases/genética , Transposases/metabolismo
4.
bioRxiv ; 2023 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-36993517

RESUMO

Traditional genome-editing reagents such as CRISPR-Cas9 achieve targeted DNA modification by introducing double-strand breaks (DSBs), thereby stimulating localized DNA repair by endogenous cellular repair factors. While highly effective at generating heterogenous knockout mutations, this approach suffers from undesirable byproducts and an inability to control product purity. Here we develop a system in human cells for programmable, DSB-free DNA integration using Type I CRISPR-associated transposons (CASTs). To adapt our previously described CAST systems, we optimized DNA targeting by the QCascade complex through a comprehensive assessment of protein design, and we developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase, TnsC, to genomic sites targeted by QCascade. After initial detection of plasmid-based transposition, we screened 15 homologous CAST systems from a wide range of bacterial hosts, identified a CAST homolog from Pseudoalteromonas that exhibited improved activity, and increased integration efficiencies through parameter optimization. We further discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, and we propose that this critical accessory factor functions to drive active disassembly of the post-transposition CAST complex, akin to its demonstrated role in Mu transposition. Our work highlights the ability to functionally reconstitute complex, multi-component machineries in human cells, and establishes a strong foundation to realize the full potential of CRISPR-associated transposons for human genome engineering.

5.
bioRxiv ; 2023 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-36993567

RESUMO

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.

6.
bioRxiv ; 2023 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-36711804

RESUMO

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.

7.
Mol Cell ; 82(3): 616-628.e5, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-35051352

RESUMO

Canonical CRISPR-Cas systems utilize RNA-guided nucleases for targeted cleavage of foreign nucleic acids, whereas some nuclease-deficient CRISPR-Cas complexes have been repurposed to direct the insertion of Tn7-like transposons. Here, we established a bioinformatic and experimental pipeline to comprehensively explore the diversity of Type I-F CRISPR-associated transposons. We report DNA integration for 20 systems and identify a highly active subset that exhibits complete orthogonality in transposon DNA mobilization. We reveal the modular nature of CRISPR-associated transposons by exploring the horizontal acquisition of targeting modules and by characterizing a system that encodes both a programmable, RNA-dependent pathway, and a fixed, RNA-independent pathway. Finally, we analyzed transposon-encoded cargo genes and found the striking presence of anti-phage defense systems, suggesting a role in transmitting innate immunity between bacteria. Collectively, this study substantially advances our biological understanding of CRISPR-associated transposon function and expands the suite of RNA-guided transposases for programmable, large-scale genome engineering.


Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , Escherichia coli/genética , Evolução Molecular , Transposases/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Escherichia coli/imunologia , Escherichia coli/metabolismo , Edição de Genes , Regulação Bacteriana da Expressão Gênica , Variação Genética , Imunidade Inata , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Transposases/metabolismo
8.
Nat Biotechnol ; 39(4): 480-489, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33230293

RESUMO

Existing technologies for site-specific integration of kilobase-sized DNA sequences in bacteria are limited by low efficiency, a reliance on recombination, the need for multiple vectors, and challenges in multiplexing. To address these shortcomings, we introduce a substantially improved version of our previously reported Tn7-like transposon from Vibrio cholerae, which uses a Type I-F CRISPR-Cas system for programmable, RNA-guided transposition. The optimized insertion of transposable elements by guide RNA-assisted targeting (INTEGRATE) system achieves highly accurate and marker-free DNA integration of up to 10 kilobases at ~100% efficiency in bacteria. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplexed insertions in three genomic loci and facile, multi-loci deletions by combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in biomedically and industrially relevant bacteria and achieved target- and species-specific integration in a complex bacterial community. This work establishes INTEGRATE as a versatile tool for multiplexed, kilobase-scale genome engineering.


Assuntos
Edição de Genes/métodos , RNA Guia de Cinetoplastídeos/genética , Vibrio cholerae/genética , Sistemas CRISPR-Cas , Elementos de DNA Transponíveis , Genoma Bacteriano , Plasmídeos/genética
9.
Nature ; 585(7825): E12, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32843756

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

10.
Nature ; 577(7789): 271-274, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31853065

RESUMO

Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements1-3. Type I CRISPR-Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase-nuclease Cas34,5, but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons6,7. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. Here we describe structures of a TniQ-Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3' end of the CRISPR RNA (crRNA). The natural Cas8-Cas5 fusion protein binds the 5' crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQ-Cascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications.


Assuntos
Sistemas CRISPR-Cas , Elementos de DNA Transponíveis , DNA Bacteriano/química , Vibrio cholerae/química , Microscopia Crioeletrônica , DNA Bacteriano/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Multimerização Proteica , Estrutura Quaternária de Proteína , RNA Bacteriano/química , Vibrio cholerae/genética
11.
Nat Biotechnol ; 37(12): 1471-1477, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31740839

RESUMO

Type I CRISPR-Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5-7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8-11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI-Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade-Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR-Cas systems can be harnessed for genome engineering applications in eukaryotic cells.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Escherichia coli , Genoma/genética , Células HEK293 , Humanos , Modelos Genéticos
12.
Nature ; 571(7764): 219-225, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31189177

RESUMO

Conventional CRISPR-Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a notable inversion of this paradigm, in which bacterial Tn7-like transposons have co-opted nuclease-deficient CRISPR-Cas systems to catalyse RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn6677 in Escherichia coli requires CRISPR- and transposon-associated molecular machineries, including a co-complex between the DNA-targeting complex Cascade and the transposition protein TniQ. Integration of donor DNA occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep-sequencing experiments reveal highly specific, genome-wide DNA insertion across dozens of unique target sites. This discovery of a fully programmable, RNA-guided integrase lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair.


Assuntos
Sistemas CRISPR-Cas/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Edição de Genes/métodos , Mutagênese Insercional/métodos , RNA Bacteriano/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Genoma Bacteriano/genética , Integrases/genética , Integrases/metabolismo , Mutagênese Sítio-Dirigida/métodos , RNA Guia de Cinetoplastídeos/genética , Especificidade por Substrato , Vibrio cholerae/genética
13.
Biosci Rep ; 38(3)2018 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-29748239

RESUMO

Since the discovery of CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-associated genes) immune systems, astonishing progress has been made on revealing their mechanistic foundations. Due to the immense potential as genome engineering tools, research has mainly focussed on a subset of Cas nucleases that target DNA. In addition, however, distinct types of RNA-targetting CRISPR-Cas systems have been identified. The focus of this review will be on the interference mechanisms of the RNA targetting type III and type VI CRISPR-Cas systems, their biological relevance and their potential for applications.


Assuntos
Bactérias/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/genética , Edição de Genes/métodos , Genoma , Imunidade Adaptativa , Animais , Bactérias/imunologia , Bacteriófagos/genética , Bacteriófagos/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , DNA/metabolismo , Elementos de DNA Transponíveis , Humanos , Plasmídeos/química , Plasmídeos/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo
14.
CRISPR J ; 1(2): 141-158, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-31021200

RESUMO

The famed physicist-turned-biologist, Max Delbrück, once remarked that, for physicists, "the field of bacterial viruses is a fine playground for serious children who ask ambitious questions." Early discoveries in that playground helped establish molecular genetics, and half a century later, biologists delving into the same field have ushered in the era of precision genome engineering. The focus has of course shifted-from bacterial viruses and their mechanisms of infection to the bacterial hosts and their mechanisms of immunity-but it is the very same evolutionary arms race that continues to awe and inspire researchers worldwide. In this review, we explore the remarkable diversity of CRISPR-Cas adaptive immune systems, describe the molecular components that mediate nucleic acid targeting, and outline the use of these RNA-guided machines for biotechnology applications. CRISPR-Cas research has yielded far more than just Cas9-based genome-editing tools, and the wide-reaching, innovative impacts of this fascinating biological playground are sure to be felt for years to come.

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