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Article ; Online: Transposons and CRISPR: Rewiring Gene Editing.

Tenjo-Castaño, Francisco / Montoya, Guillermo / Carabias, Arturo

2022 Volume 62, Issue 24, Page(s) 3521–3532

Abstract: 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 ... ...

Abstract	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 transposon-associated 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.
MeSH term(s)	Humans ; Gene Editing ; CRISPR-Cas Systems ; Endonucleases/genetics ; DNA ; RNA
Chemical Substances	Endonucleases (EC 3.1.-) ; DNA (9007-49-2) ; RNA (63231-63-0)
Language	English
Publishing date	2022-09-21
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021/acs.biochem.2c00379
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Article: C3G self-regulatory mechanism revealed: implications for hematopoietic malignancies.

Carabias, Arturo / Guerrero, Carmen / de Pereda, José M

Molecular & cellular oncology

2020 Volume 8, Issue 1, Page(s) 1837581

Abstract: Abnormally increased signaling by the GTPase RAP1 favors progression of diverse tumors. We have characterized the auto-regulation and activation of C3G (RAPGEF1), an activator of RAP1. This led us to discover mutations in non-Hodgkin's lymphomas that ... ...

Abstract	Abnormally increased signaling by the GTPase RAP1 favors progression of diverse tumors. We have characterized the auto-regulation and activation of C3G (RAPGEF1), an activator of RAP1. This led us to discover mutations in non-Hodgkin's lymphomas that activate C3G-RAP1 constitutively, suggesting that deregulation of C3G may favor the dissemination of tumor cells.
Language	English
Publishing date	2020-12-01
Publishing country	United States
Document type	Journal Article
ISSN	2372-3556
ISSN	2372-3556
DOI	10.1080/23723556.2020.1837581
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Article ; Online: Pathogen-specific structural features of

Manso, José A / Carabias, Arturo / Sárkány, Zsuzsa / de Pereda, José M / Pereira, Pedro José Barbosa / Macedo-Ribeiro, Sandra

mBio

2023 Volume 14, Issue 4, Page(s) e0063823

Abstract: An important feature associated ... ...

Abstract	An important feature associated with
MeSH term(s)	Humans ; Candida albicans ; Antifungal Agents/pharmacology ; Antifungal Agents/metabolism ; Candidiasis/microbiology ; Signal Transduction ; Guanine Nucleotide Exchange Factors/metabolism ; Fungal Proteins/genetics ; Fungal Proteins/metabolism ; Hyphae
Chemical Substances	Antifungal Agents ; Guanine Nucleotide Exchange Factors ; Fungal Proteins
Language	English
Publishing date	2023-08-01
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2557172-2
ISSN	2150-7511 ; 2161-2129
ISSN (online)	2150-7511
ISSN	2161-2129
DOI	10.1128/mbio.00638-23
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Article: Fluence requirements in existing UV disinfection facilities to comply with EU validation performance targets for reclaimed water: a case study.

Verónica Carabias, Amaia / Gil, Maria Isabel / Pérez, Juan Marcos / Abellán, Manuel / Rancaño, Amador / José Simón, Pedro / Truchado, Pilar / Allende, Ana

Water science and technology : a journal of the International Association on Water Pollution Research

2023 Volume 88, Issue 4, Page(s) 1131–1141

Abstract: Since 2020, there is a new European Regulation (EU, 2020/741) on minimum requirements for water reuse, where routine and validation monitoring requirements (log reductions of indicator microorganisms and reference pathogens) have been established. Many ... ...

Abstract	Since 2020, there is a new European Regulation (EU, 2020/741) on minimum requirements for water reuse, where routine and validation monitoring requirements (log reductions of indicator microorganisms and reference pathogens) have been established. Many reclamation facilities that are already in operation might have difficulties to comply with these performance targets. Existing disinfection systems must be expanded and upgraded. In the case of UV disinfection systems, fluence requirements must be determined to properly design with a focus on the safety and economic-environmental viability of reclaimed water. This study can be used as a reference to develop fluence-response curves for Clostridium perfringens spores, Escherichia coli, and total and F-specific coliphages, indicator microorganisms referred to in the new European Regulation. Eight UV-LED collimated beam tests were performed. Samples were obtained from filtered effluent of secondary treatment from two wastewater treatment plants (WWTPs) which ranged between 30 and 54%. Results showed UV sensitivity of 33.46 mJ/cm
MeSH term(s)	Disinfection ; Escherichia coli ; Water
Chemical Substances	Water (059QF0KO0R)
Language	English
Publishing date	2023-08-31
Publishing country	England
Document type	Journal Article
ZDB-ID	764273-8
ISSN	1996-9732 ; 0273-1223
ISSN (online)	1996-9732
ISSN	0273-1223
DOI	10.2166/wst.2023.258
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Article ; Online: TnpB structure reveals minimal functional core of Cas12 nuclease family.

Sasnauskas, Giedrius / Tamulaitiene, Giedre / Druteika, Gytis / Carabias, Arturo / Silanskas, Arunas / Kazlauskas, Darius / Venclovas, Česlovas / Montoya, Guillermo / Karvelis, Tautvydas / Siksnys, Virginijus

Nature

2023 Volume 616, Issue 7956, Page(s) 384–389

Abstract: The widespread TnpB proteins of IS200/IS605 transposon family have recently emerged as the smallest RNA-guided nucleases capable of targeted genome editing in eukaryotic ... ...

Abstract	The widespread TnpB proteins of IS200/IS605 transposon family have recently emerged as the smallest RNA-guided nucleases capable of targeted genome editing in eukaryotic cells
MeSH term(s)	CRISPR-Associated Proteins/chemistry ; CRISPR-Associated Proteins/classification ; CRISPR-Associated Proteins/metabolism ; CRISPR-Associated Proteins/ultrastructure ; CRISPR-Cas Systems/genetics ; Cryoelectron Microscopy ; Deinococcus/enzymology ; Deinococcus/genetics ; DNA/chemistry ; DNA/genetics ; DNA/metabolism ; DNA/ultrastructure ; DNA Transposable Elements/genetics ; Endonucleases/chemistry ; Endonucleases/classification ; Endonucleases/metabolism ; Endonucleases/ultrastructure ; Evolution, Molecular ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems
Chemical Substances	CRISPR-Associated Proteins ; DNA (9007-49-2) ; DNA Transposable Elements ; Endonucleases (EC 3.1.-) ; RNA, Guide, CRISPR-Cas Systems
Language	English
Publishing date	2023-04-05
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	120714-3
ISSN	1476-4687 ; 0028-0836
ISSN (online)	1476-4687
ISSN	0028-0836
DOI	10.1038/s41586-023-05826-x
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Article ; Online: Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms.

Rodríguez-Blázquez, Antonio / Carabias, Arturo / Morán-Vaquero, Alba / de Cima, Sergio / Luque-Ortega, Juan R / Alfonso, Carlos / Schuck, Peter / Manso, José Antonio / Macedo-Ribeiro, Sandra / Guerrero, Carmen / de Pereda, José M

Cell communication and signaling : CCS

2023 Volume 21, Issue 1, Page(s) 30

Abstract: Background: C3G is a guanine nucleotide exchange factor (GEF) that activates Rap1 to promote cell adhesion. Resting C3G is autoinhibited and the GEF activity is released by stimuli that signal through tyrosine kinases. C3G is activated by tyrosine ... ...

Abstract	Background: C3G is a guanine nucleotide exchange factor (GEF) that activates Rap1 to promote cell adhesion. Resting C3G is autoinhibited and the GEF activity is released by stimuli that signal through tyrosine kinases. C3G is activated by tyrosine phosphorylation and interaction with Crk adaptor proteins, whose expression is elevated in multiple human cancers. However, the molecular details of C3G activation and the interplay between phosphorylation and Crk interaction are poorly understood. Methods: We combined biochemical, biophysical, and cell biology approaches to elucidate the mechanisms of C3G activation. Binding of Crk adaptor proteins to four proline-rich motifs (P1 to P4) in C3G was characterized in vitro using isothermal titration calorimetry and sedimentation velocity, and in Jurkat and HEK293T cells by affinity pull-down assays. The nucleotide exchange activity of C3G over Rap1 was measured using nucleotide-dissociation kinetic assays. Jurkat cells were also used to analyze C3G translocation to the plasma membrane and the C3G-dependent activation of Rap1 upon ligation of T cell receptors. Results: CrkL interacts through its SH3N domain with sites P1 and P2 of inactive C3G in vitro and in Jurkat and HEK293T cells, and these sites are necessary to recruit C3G to the plasma membrane. However, direct stimulation of the GEF activity requires binding of Crk proteins to the P3 and P4 sites. P3 is occluded in resting C3G and is essential for activation, while P4 contributes secondarily towards complete stimulation. Tyrosine phosphorylation of C3G alone causes marginal activation. Instead, phosphorylation primes C3G lowering the concentration of Crk proteins required for activation and increasing the maximum activity. Unexpectedly, optimal activation also requires the interaction of CrkL-SH2 domain with phosphorylated C3G. Conclusion: Our study revealed that phosphorylation of C3G by Src and Crk-binding form a two-factor mechanism that ensures tight control of C3G activation. Additionally, the simultaneous SH2 and SH3N interaction of CrkL with C3G, required for the activation, reveals a novel adaptor-independent function of Crk proteins relevant to understanding their role in physiological signaling and their deregulation in diseases. Video abstract.
MeSH term(s)	Humans ; Guanine Nucleotide Exchange Factors/metabolism ; Guanine Nucleotide-Releasing Factor 2/metabolism ; HEK293 Cells ; Nuclear Proteins/metabolism ; Nucleotides/metabolism ; Proto-Oncogene Proteins c-crk/metabolism ; src Homology Domains ; Tyrosine/metabolism
Chemical Substances	CRK protein, human ; Guanine Nucleotide Exchange Factors ; Guanine Nucleotide-Releasing Factor 2 ; Nuclear Proteins ; Nucleotides ; Proto-Oncogene Proteins c-crk ; Tyrosine (42HK56048U) ; TERF2IP protein, human
Language	English
Publishing date	2023-02-03
Publishing country	England
Document type	Video-Audio Media ; Journal Article ; Research Support, N.I.H., Intramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2126315-2
ISSN	1478-811X ; 1478-811X
ISSN (online)	1478-811X
ISSN	1478-811X
DOI	10.1186/s12964-023-01042-2
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Article ; Online: Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3.

Carabias, Arturo / Fuglsang, Anders / Temperini, Piero / Pape, Tillmann / Sofos, Nicholas / Stella, Stefano / Erlendsson, Simon / Montoya, Guillermo

Nature communications

2021 Volume 12, Issue 1, Page(s) 4476

Abstract: CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into ... ...

Abstract	CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.
MeSH term(s)	Bacteriophages/enzymology ; Bacteriophages/genetics ; CRISPR-Associated Proteins/chemistry ; CRISPR-Associated Proteins/genetics ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; Catalytic Domain ; Cryoelectron Microscopy ; DNA Cleavage ; Endodeoxyribonucleases/chemistry ; Endodeoxyribonucleases/genetics ; Endodeoxyribonucleases/metabolism ; Escherichia coli Proteins/chemistry ; Escherichia coli Proteins/genetics ; Escherichia coli Proteins/metabolism ; Gene Editing ; Models, Molecular ; Mutagenesis, Site-Directed ; Protein Conformation ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism ; RNA, Viral/genetics ; RNA, Viral/metabolism
Chemical Substances	CRISPR-Associated Proteins ; Escherichia coli Proteins ; RNA, Guide, CRISPR-Cas Systems ; RNA, Viral ; ruvC protein, E coli ; Endodeoxyribonucleases (EC 3.1.-)
Language	English
Publishing date	2021-07-22
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-021-24707-3
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Article: Giant retroperitoneal ganglioneuroma.

Acín-Gándara, D / Carabias, A / Bertomeu, A / Giménez-Alvira, L / Colao, L / Limones, M

Revista espanola de enfermedades digestivas : organo oficial de la Sociedad Espanola de Patologia Digestiva

2010 Volume 102, Issue 3, Page(s) 205–207

MeSH term(s)	Adult ; Female ; Ganglioneuroma/diagnosis ; Ganglioneuroma/surgery ; Humans ; Pregnancy ; Pregnancy Complications, Neoplastic/diagnosis ; Pregnancy Complications, Neoplastic/surgery ; Retroperitoneal Neoplasms/diagnosis ; Retroperitoneal Neoplasms/surgery
Language	Spanish
Publishing date	2010-04-01
Publishing country	Spain
Document type	Case Reports ; Journal Article
ZDB-ID	1070381-0
ISSN	1130-0108 ; 0212-7512
ISSN	1130-0108 ; 0212-7512
DOI	10.4321/s1130-01082010000300008
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Article ; Online: Mechanisms of autoregulation of C3G, activator of the GTPase Rap1, and its catalytic deregulation in lymphomas.

Carabias, Arturo / Gómez-Hernández, María / de Cima, Sergio / Rodríguez-Blázquez, Antonio / Morán-Vaquero, Alba / González-Sáenz, Patricia / Guerrero, Carmen / de Pereda, José M

Science signaling

2020 Volume 13, Issue 647

Abstract: C3G is a guanine nucleotide exchange factor (GEF) that regulates cell adhesion and migration by activating the GTPase Rap1. The GEF activity of C3G is stimulated by the adaptor proteins Crk and CrkL and by tyrosine phosphorylation. Here, we uncovered ... ...

Abstract	C3G is a guanine nucleotide exchange factor (GEF) that regulates cell adhesion and migration by activating the GTPase Rap1. The GEF activity of C3G is stimulated by the adaptor proteins Crk and CrkL and by tyrosine phosphorylation. Here, we uncovered mechanisms of C3G autoinhibition and activation. Specifically, we found that two intramolecular interactions regulate the activity of C3G. First, an autoinhibitory region (AIR) within the central domain of C3G binds to and blocks the catalytic Cdc25H domain. Second, the binding of the protein's N-terminal domain to its Ras exchanger motif (REM) is required for its GEF activity. CrkL activated C3G by displacing the AIR/Cdc25HD interaction. Two missense mutations in the AIR found in non-Hodgkin's lymphomas, Y554H and M555K, disrupted the autoinhibitory mechanism. Expression of C3G-Y554H or C3G-M555K in Ba/F3 pro-B cells caused constitutive activation of Rap1 and, consequently, the integrin LFA-1. Our findings suggest that sustained Rap1 activation by deregulated C3G might promote progression of lymphomas and that designing therapeutics to target C3G might treat these malignancies.
MeSH term(s)	Adaptor Proteins, Signal Transducing/metabolism ; Amino Acid Sequence ; Animals ; Biocatalysis ; COS Cells ; Cell Line ; Chlorocebus aethiops ; Guanine Nucleotide Exchange Factors/chemistry ; Guanine Nucleotide Exchange Factors/genetics ; Guanine Nucleotide Exchange Factors/metabolism ; HEK293 Cells ; Homeostasis/physiology ; Humans ; Lymphoma, Non-Hodgkin/genetics ; Lymphoma, Non-Hodgkin/metabolism ; Mice ; Mutation ; Protein Binding ; Sequence Homology, Amino Acid ; rap1 GTP-Binding Proteins/genetics ; rap1 GTP-Binding Proteins/metabolism ; src Homology Domains
Chemical Substances	Adaptor Proteins, Signal Transducing ; CRKL protein ; Guanine Nucleotide Exchange Factors ; rap1 GTP-Binding Proteins (EC 3.6.5.2)
Language	English
Publishing date	2020-09-01
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2417226-1
ISSN	1937-9145 ; 1945-0877
ISSN (online)	1937-9145
ISSN	1945-0877
DOI	10.1126/scisignal.abb7075
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Article ; Online: Textbook outcome in distal pancreatectomy: A multicenter study.

Villodre, Celia / Del Río-Martín, Juan / Blanco-Fernández, Gerardo / Cantalejo-Díaz, Miguel / Pardo, Fernando / Carbonell, Silvia / Muñoz-Forner, Elena / Carabias, Alberto / Manuel-Vazquez, Alba / Hernández-Rivera, Pedro J / Jaén-Torrejimeno, Isabel / Kälviäinen-Mejia, Helga K / Rotellar, Fernando / Garcés-Albir, Marina / Latorre, Raquel / Longoria-Dubocq, Texell / De Armas-Conde, Noelia / Serrablo, Alejandro / Esteban Gordillo, Sara /

Sabater, Luis / Serradilla-Martín, Mario / Ramia, José M

Surgery

2023 Volume 175, Issue 4, Page(s) 1134–1139

Abstract: Background: Textbook outcome is an interesting quality metrics tool. Information on textbook outcomes in distal pancreatectomy is very scarce. In this study we determined textbook outcome in a distal pancreatectomy multicenter database and propose a ... ...

Abstract	Background: Textbook outcome is an interesting quality metrics tool. Information on textbook outcomes in distal pancreatectomy is very scarce. In this study we determined textbook outcome in a distal pancreatectomy multicenter database and propose a specific definition of textbook outcome-distal pancreatectomy that includes pancreatic fistula. Methods: Retrospective multicenter observational study of distal pancreatectomy performed at 8 hepatopancreatobiliary surgery units from January 1, 2008, to December 31, 2018. The inclusion criteria were any scheduled distal pancreatectomy performed for any diagnosis and age > 18 years. Specific textbook outcome-distal pancreatectomy was defined as hospital stay P < 75, no Clavien-Dindo complications (≥ III), no hospital mortality, and no readmission recorded at 90 days, and the absence of pancreatic fistula (B/C). Results: Of the 450 patients included, 262 (58.2%) obtained textbook outcomes. Prolonged stay was the parameter most frequently associated with failure to achieve textbook outcomes. The textbook outcome group presented the following results. Preoperative: lower American Society of Anesthesiologists score < III, a lower percentage of smokers, and less frequent tumor invasion of neighboring organs or vascular invasion; operative: major laparoscopic approach, and less resection of neighboring organs and less operative transfusion; postoperative: lower percentage of delayed gastric emptying and pancreatic fistula B/C, and diagnosis other an adenocarcinoma. In the multivariate study, the American Society of Anesthesiologists score > II, resection of neighboring organs, B/C pancreatic fistula, and delayed gastric emptying were associated with failure to achieve textbook outcomes. Conclusion: The textbook outcome rate in our 450 pancreaticoduodenectomies was 58.2%. In the multivariate analysis, the causes of failure to achieve textbook outcomes were American Society of Anesthesiologists score > II, resection of neighboring organs, pancreatic fistula B/C, and delayed gastric emptying. We believe that pancreatic fistula should be added to the specific definition of textbook outcome-distal pancreatectomy because it is the most frequent complication of this procedure.
MeSH term(s)	Humans ; Adult ; Middle Aged ; Pancreatic Fistula/epidemiology ; Pancreatic Fistula/etiology ; Pancreatic Fistula/surgery ; Pancreatic Neoplasms ; Pancreatectomy/methods ; Gastroparesis ; Retrospective Studies ; Postoperative Complications/epidemiology ; Postoperative Complications/etiology ; Postoperative Complications/diagnosis ; Treatment Outcome ; Laparoscopy/adverse effects
Language	English
Publishing date	2023-12-09
Publishing country	United States
Document type	Multicenter Study ; Observational Study ; Journal Article
ZDB-ID	202467-6
ISSN	1532-7361 ; 0039-6060
ISSN (online)	1532-7361
ISSN	0039-6060
DOI	10.1016/j.surg.2023.11.012
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