36 results on '"Brouns, Stan J.J."'
Search Results
2. Modulating CRISPR-Cas Genome Editing Using Guide-Complementary DNA Oligonucleotides
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Swartjes, Thomas, Shang, Peng, Van Den Berg, Dennis T.M., Künne, Tim, Geijsen, Niels, Brouns, Stan J.J., van der Oost, John, Staals, Raymond H.J., Notebaart, Richard A., Swartjes, Thomas, Shang, Peng, Van Den Berg, Dennis T.M., Künne, Tim, Geijsen, Niels, Brouns, Stan J.J., van der Oost, John, Staals, Raymond H.J., and Notebaart, Richard A.
- Abstract
Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) has revolutionized genome editing and has great potential for many applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. In this study, we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides inhibit Cas9 activity in human cells, reducing both on- A nd off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: The length of the complementary region and the presence of a protospacer adjacent motif-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.
- Published
- 2022
3. Adaptation by Type V-A and V-B CRISPR-Cas Systems Demonstrates Conserved Protospacer Selection Mechanisms Between Diverse CRISPR-Cas Types
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Wu, Wen Y., Jackson, Simon A., Almendros, Cristóbal, Haagsma, Anna C., Yilmaz, Suzan, Gort, Gerrit, van der Oost, John, Brouns, Stan J.J., Staals, Raymond H.J., Wu, Wen Y., Jackson, Simon A., Almendros, Cristóbal, Haagsma, Anna C., Yilmaz, Suzan, Gort, Gerrit, van der Oost, John, Brouns, Stan J.J., and Staals, Raymond H.J.
- Abstract
Adaptation of clustered regularly interspaced short palindromic repeats (CRISPR) arrays is a crucial process responsible for the unique, adaptive nature of CRISPR-Cas immune systems. The acquisition of new CRISPR spacers from mobile genetic elements has previously been studied for several types of CRISPR-Cas systems. In this study, we used a high-throughput sequencing approach to characterize CRISPR adaptation of the type V-A system from Francisella novicida and the type V-B system from Alicyclobacillus acidoterrestris. In contrast to other class 2 CRISPR-Cas systems, we found that for the type V-A and V-B systems, the Cas12 nucleases are dispensable for spacer acquisition, with only Cas1 and Cas2 (type V-A) or Cas4/1 and Cas2 (type V-B) being necessary and sufficient. Whereas the catalytic activity of Cas4 is not essential for adaptation, Cas4 activity is required for correct protospacer adjacent motif selection in both systems and for prespacer trimming in type V-A. In addition, we provide evidence for acquisition of RecBCD-produced DNA fragments by both systems, but with spacers derived from foreign DNA being incorporated preferentially over those derived from the host chromosome. Our work shows that several spacer acquisition mechanisms are conserved between diverse CRISPR-Cas systems, but also highlights unexpected nuances between similar systems that generally contribute to a bias of gaining immunity against invading genetic elements.
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- 2022
4. Short prokaryotic Argonaute systems trigger cell death upon detection of invading DNA
- Author
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Koopal, Balwina, Potocnik, Ana, Mutte, Sumanth K., Aparicio-Maldonado, Cristian, Lindhoud, Simon, Vervoort, Jacques J.M., Brouns, Stan J.J., Swarts, Daan C., Koopal, Balwina, Potocnik, Ana, Mutte, Sumanth K., Aparicio-Maldonado, Cristian, Lindhoud, Simon, Vervoort, Jacques J.M., Brouns, Stan J.J., and Swarts, Daan C.
- Abstract
Argonaute proteins use single-stranded RNA or DNA guides to target complementary nucleic acids. This allows eukaryotic Argonaute proteins to mediate RNA interference and long prokaryotic Argonaute proteins to interfere with invading nucleic acids. The function and mechanisms of the phylogenetically distinct short prokaryotic Argonaute proteins remain poorly understood. We demonstrate that short prokaryotic Argonaute and the associated TIR-APAZ (SPARTA) proteins form heterodimeric complexes. Upon guide RNA-mediated target DNA binding, four SPARTA heterodimers form oligomers in which TIR domain-mediated NAD(P)ase activity is unleashed. When expressed in Escherichia coli, SPARTA is activated in the presence of highly transcribed multicopy plasmid DNA, which causes cell death through NAD(P)+ depletion. This results in the removal of plasmid-invaded cells from bacterial cultures. Furthermore, we show that SPARTA can be repurposed for the programmable detection of DNA sequences. In conclusion, our work identifies SPARTA as a prokaryotic immune system that reduces cell viability upon RNA-guided detection of invading DNA.
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- 2022
5. Single cell variability of CRISPR-Cas interference and adaptation
- Author
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McKenzie, Rebecca E., Keizer, Emma M., Vink, Jochem N.A., van Lopik, Jasper, Büke, Ferhat, Kalkman, Vera, Fleck, Christian, Tans, Sander J., Brouns, Stan J.J., McKenzie, Rebecca E., Keizer, Emma M., Vink, Jochem N.A., van Lopik, Jasper, Büke, Ferhat, Kalkman, Vera, Fleck, Christian, Tans, Sander J., and Brouns, Stan J.J.
- Abstract
While CRISPR-Cas defence mechanisms have been studied on a population level, their temporal dynamics and variability in individual cells have remained unknown. Using a microfluidic device, time-lapse microscopy and mathematical modelling, we studied invader clearance in Escherichia coli across multiple generations. We observed that CRISPR interference is fast with a narrow distribution of clearance times. In contrast, for invaders with escaping PAM mutations we found large cell-to-cell variability, which originates from primed CRISPR adaptation. Faster growth and cell division and higher levels of Cascade increase the chance of clearance by interference, while slower growth is associated with increased chances of clearance by priming. Our findings suggest that Cascade binding to the mutated invader DNA, rather than spacer integration, is the main source of priming heterogeneity. The highly stochastic nature of primed CRISPR adaptation implies that only subpopulations of bacteria are able to respond quickly to invading threats. We conjecture that CRISPR-Cas dynamics and heterogeneity at the cellular level are crucial to understanding the strategy of bacteria in their competition with other species and phages.
- Published
- 2022
6. assessment of Cas9-driven genome editing in presence of DNA oligos in CML cells
- Author
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Swartjes, Thomas, Shang, Peng, Van Den Berg, Dennis T.M., Künne, Tim, Geijsen, Niels, Brouns, Stan J.J., van der Oost, John, Staals, Raymond H.J., Notebaart, Richard A., Swartjes, Thomas, Shang, Peng, Van Den Berg, Dennis T.M., Künne, Tim, Geijsen, Niels, Brouns, Stan J.J., van der Oost, John, Staals, Raymond H.J., and Notebaart, Richard A.
- Abstract
We assessed indels resulting from Cas9-induced genome editing in CML cells in the presence of guide-complementary DNA oligonucleotides intended to inhibit Cas9 activity. We amplified pre-determined loci that were target with the supplied guides or were expected to be off-target sites for these guides. The on-target loci are EMX1 and FANCF., We assessed indels resulting from Cas9-induced genome editing in CML cells in the presence of guide-complementary DNA oligonucleotides intended to inhibit Cas9 activity. We amplified pre-determined loci that were target with the supplied guides or were expected to be off-target sites for these guides. The on-target loci are EMX1 and FANCF.
- Published
- 2022
7. SPARTA-related RNA and DNA
- Author
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Koopal, Balwina, Potocnik, Ana, Mutte, Sumanth K., Aparicio-Maldonado, Cristian, Lindhoud, Simon, Vervoort, Jacques J.M., Brouns, Stan J.J., Swarts, Daan C., Koopal, Balwina, Potocnik, Ana, Mutte, Sumanth K., Aparicio-Maldonado, Cristian, Lindhoud, Simon, Vervoort, Jacques J.M., Brouns, Stan J.J., and Swarts, Daan C.
- Abstract
SPARTA-related small RNA, long RNA, and DNA Overall design: Total and small RNAseq data of total RNA from cells expressing SPARTA or co-purified with SPARTA complexes expressed in E. coli BL21(DE3), SPARTA-related small RNA, long RNA, and DNA Overall design: Total and small RNAseq data of total RNA from cells expressing SPARTA or co-purified with SPARTA complexes expressed in E. coli BL21(DE3)
- Published
- 2021
8. Direct Visualization of Native CRISPR Target Search in Live Bacteria Reveals Cascade DNA Surveillance Mechanism
- Author
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Vink, Jochem N.A., Martens, Koen J.A., Vlot, Marnix, McKenzie, Rebecca E., Almendros, Cristóbal, Estrada Bonilla, Boris, Brocken, Daan J.W., Hohlbein, Johannes, Brouns, Stan J.J., Vink, Jochem N.A., Martens, Koen J.A., Vlot, Marnix, McKenzie, Rebecca E., Almendros, Cristóbal, Estrada Bonilla, Boris, Brocken, Daan J.W., Hohlbein, Johannes, and Brouns, Stan J.J.
- Abstract
Vink et al. tracked single CRISPR RNA-surveillance complexes (Cascade) in the native host cell and determined the influence of Cascade copy numbers, PAM scanning speed, and the presence of CRISPR arrays and transcription on their ability to find and clear invading mobile genetic elements from the cell.
- Published
- 2020
9. Extracting Transition Rates in Particle Tracking Using Analytical Diffusion Distribution Analysis
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Vink, Jochem N.A., Brouns, Stan J.J., Hohlbein, Johannes, Vink, Jochem N.A., Brouns, Stan J.J., and Hohlbein, Johannes
- Abstract
Single-particle tracking is an important technique in the life sciences to understand the kinetics of biomolecules. The analysis of apparent diffusion coefficients in vivo, for example, enables researchers to determine whether biomolecules are moving alone, as part of a larger complex, or are bound to large cellular components such as the membrane or chromosomal DNA. A remaining challenge has been to retrieve quantitative kinetic models, especially for molecules that rapidly switch between different diffusional states. Here, we present analytical diffusion distribution analysis (anaDDA), a framework that allows for extracting transition rates from distributions of apparent diffusion coefficients calculated from short trajectories that feature less than 10 localizations per track. Under the assumption that the system is Markovian and diffusion is purely Brownian, we show that theoretically predicted distributions accurately match simulated distributions and that anaDDA outperforms existing methods to retrieve kinetics, especially in the fast regime of 0.1–10 transitions per imaging frame. AnaDDA does account for the effects of confinement and tracking window boundaries. Furthermore, we added the option to perform global fitting of data acquired at different frame times to allow complex models with multiple states to be fitted confidently. Previously, we have started to develop anaDDA to investigate the target search of CRISPR-Cas complexes. In this work, we have optimized the algorithms and reanalyzed experimental data of DNA polymerase I diffusing in live Escherichia coli. We found that long-lived DNA interaction by DNA polymerase are more abundant upon DNA damage, suggesting roles in DNA repair. We further revealed and quantified fast DNA probing interactions that last shorter than 10 ms. AnaDDA pushes the boundaries of the timescale of interactions that can be probed with single-particle tracking and is a mathematically rigorous framework that can be further expan
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- 2020
10. Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants
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Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Makarova, Kira S., Wolf, Yuri I., Iranzo, Jaime, Shmakov, Sergey A., Alkhnbashi, Omer S., Brouns, Stan J.J., Charpentier, Emmanuelle, Cheng, David, Haft, Daniel H., Horvath, Philippe, Moineau, Sylvain, Mojica, Francisco J.M., Scott, David, Shah, Shiraz A., Siksnys, Virginijus, Terns, Michael P., Venclovas, Česlovas, White, Malcolm F., Yakunin, Alexander F., Yan, Winston, Zhang, Feng, Garrett, Roger A., Backofen, Rolf, Oost, John van der, Barrangou, Rodolphe, Koonin, Eugene V., Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Makarova, Kira S., Wolf, Yuri I., Iranzo, Jaime, Shmakov, Sergey A., Alkhnbashi, Omer S., Brouns, Stan J.J., Charpentier, Emmanuelle, Cheng, David, Haft, Daniel H., Horvath, Philippe, Moineau, Sylvain, Mojica, Francisco J.M., Scott, David, Shah, Shiraz A., Siksnys, Virginijus, Terns, Michael P., Venclovas, Česlovas, White, Malcolm F., Yakunin, Alexander F., Yan, Winston, Zhang, Feng, Garrett, Roger A., Backofen, Rolf, Oost, John van der, Barrangou, Rodolphe, and Koonin, Eugene V.
- Abstract
The number and diversity of known CRISPR–Cas systems have substantially increased in recent years. Here, we provide an updated evolutionary classification of CRISPR–Cas systems and cas genes, with an emphasis on the major developments that have occurred since the publication of the latest classification, in 2015. The new classification includes 2 classes, 6 types and 33 subtypes, compared with 5 types and 16 subtypes in 2015. A key development is the ongoing discovery of multiple, novel class 2 CRISPR–Cas systems, which now include 3 types and 17 subtypes. A second major novelty is the discovery of numerous derived CRISPR–Cas variants, often associated with mobile genetic elements that lack the nucleases required for interference. Some of these variants are involved in RNA-guided transposition, whereas others are predicted to perform functions distinct from adaptive immunity that remain to be characterized experimentally. The third highlight is the discovery of numerous families of ancillary CRISPR-linked genes, often implicated in signal transduction. Together, these findings substantially clarify the functional diversity and evolutionary history of CRISPR–Cas.
- Published
- 2019
11. Global phylogeography and ancient evolution of the widespread human gut virus crAssphage
- Author
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Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Edwards, Robert A., Vega, Alejandro A., Norman, Holly M., Ohaeri, Maria, Levi, Kyle, Dinsdale, Elizabeth A., Cinek, Ondrej, Aziz, Ramy K., McNair, Katelyn, Barr, Jeremy J., Bibby, Kyle, Shimashita, John, Stachler, Elyse N., Stene, Lars C., Strain, Ronan, Stumpf, Rebecca, Torres, Pedro J., Twaddle, Alan, Ugochi Ibekwe, MaryAnn, Villagra, Nicolás, Wandro, Stephen, Brouns, Stan J.J., White, Bryan, Whiteley, Andy, Whiteson, Katrine L., Wijmenga, Cisca, Zambrano, Maria M., Zschach, Henrike, Dutilh, Bas E., Cazares, Adrian, Jonge, Patrick A. de, Desnues, Christelle, Díaz Muñoz, Samuel L., Fineran, Peter C., Kurilshikov, Alexander, Lavigne, Rob, Mazankova, Karla, McCarthy, David T., Nobrega, Franklin L., Reyes Muñoz, Alejandro, Tapia, German, Trefault, Nicole, Tyakht, Alexander V., Vinuesa, Pablo, Wagemans, Jeroen, Zhernakova, Alexandra, Aarestrup, Frank M., Ahmadov, Gunduz, Alassaf, Abeer, Anton, Josefa, Asangba, Abigail, Billings, Emma K., Cantu, Vito Adrian, Carlton, Jane M., Cazares, Daniel, Cho, Gyu-Sung, Condeff, Tess, Cortés, Pilar, Cranfield, Mike, Cuevas, Daniel A., De la Iglesia, Rodrigo, Decewicz, Przemyslaw, Doane, Michael P., Dominy, Nathaniel J., Dziewit, Lukasz, Elwasila, Bashir Mukhtar, Eren, A. Murat, Franz, Charles, Fu, Jingyuan, Garcia-Aljaro, Cristina, Ghedin, Elodie, Gulino, Kristen M., Haggerty, John M., Head, Steven R., Hendriksen, Rene S., Hill, Colin, Hyöty, Heikki, Ilina, Elena N., Irwin, Mitchell T., Jeffries, Thomas C., Jofre, Juan, Junge, Randall E., Kelley, Scott T., Mirzaei, Mohammadali Khan, Kowalewski, Martin, Kumaresan, Deepak, Leigh, Steven R., Lipson, David, Lisitsyna, Eugenia S., Llagostera, Montserrat, Maritz, Julia M., Marr, Linsey C., McCann, Angela, Molshanski-Mor, Shahar, Monteiro, Silvia, Moreira-Grez, Benjamin, Morris, Megan, Mugisha, Lawrence, Muniesa, Maite, Neve, Horst, Nguyen, Nam-phuong, Nigro, Olivia D., Nilsson, Anders S., O’Connell, Taylor, Odeh, Rasha, Oliver, Andrew, Piuri, Mariana, Prussin II, Aaron J., Qimron, Udi, Quan, Zhe-Xue, Rainetova, Petra, Ramírez-Rojas, Adán, Raya, Raul, Reasor, Kim, Rice, Gillian A.O., Rossi, Alessandro, Santos, Ricardo, Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Edwards, Robert A., Vega, Alejandro A., Norman, Holly M., Ohaeri, Maria, Levi, Kyle, Dinsdale, Elizabeth A., Cinek, Ondrej, Aziz, Ramy K., McNair, Katelyn, Barr, Jeremy J., Bibby, Kyle, Shimashita, John, Stachler, Elyse N., Stene, Lars C., Strain, Ronan, Stumpf, Rebecca, Torres, Pedro J., Twaddle, Alan, Ugochi Ibekwe, MaryAnn, Villagra, Nicolás, Wandro, Stephen, Brouns, Stan J.J., White, Bryan, Whiteley, Andy, Whiteson, Katrine L., Wijmenga, Cisca, Zambrano, Maria M., Zschach, Henrike, Dutilh, Bas E., Cazares, Adrian, Jonge, Patrick A. de, Desnues, Christelle, Díaz Muñoz, Samuel L., Fineran, Peter C., Kurilshikov, Alexander, Lavigne, Rob, Mazankova, Karla, McCarthy, David T., Nobrega, Franklin L., Reyes Muñoz, Alejandro, Tapia, German, Trefault, Nicole, Tyakht, Alexander V., Vinuesa, Pablo, Wagemans, Jeroen, Zhernakova, Alexandra, Aarestrup, Frank M., Ahmadov, Gunduz, Alassaf, Abeer, Anton, Josefa, Asangba, Abigail, Billings, Emma K., Cantu, Vito Adrian, Carlton, Jane M., Cazares, Daniel, Cho, Gyu-Sung, Condeff, Tess, Cortés, Pilar, Cranfield, Mike, Cuevas, Daniel A., De la Iglesia, Rodrigo, Decewicz, Przemyslaw, Doane, Michael P., Dominy, Nathaniel J., Dziewit, Lukasz, Elwasila, Bashir Mukhtar, Eren, A. Murat, Franz, Charles, Fu, Jingyuan, Garcia-Aljaro, Cristina, Ghedin, Elodie, Gulino, Kristen M., Haggerty, John M., Head, Steven R., Hendriksen, Rene S., Hill, Colin, Hyöty, Heikki, Ilina, Elena N., Irwin, Mitchell T., Jeffries, Thomas C., Jofre, Juan, Junge, Randall E., Kelley, Scott T., Mirzaei, Mohammadali Khan, Kowalewski, Martin, Kumaresan, Deepak, Leigh, Steven R., Lipson, David, Lisitsyna, Eugenia S., Llagostera, Montserrat, Maritz, Julia M., Marr, Linsey C., McCann, Angela, Molshanski-Mor, Shahar, Monteiro, Silvia, Moreira-Grez, Benjamin, Morris, Megan, Mugisha, Lawrence, Muniesa, Maite, Neve, Horst, Nguyen, Nam-phuong, Nigro, Olivia D., Nilsson, Anders S., O’Connell, Taylor, Odeh, Rasha, Oliver, Andrew, Piuri, Mariana, Prussin II, Aaron J., Qimron, Udi, Quan, Zhe-Xue, Rainetova, Petra, Ramírez-Rojas, Adán, Raya, Raul, Reasor, Kim, Rice, Gillian A.O., Rossi, Alessandro, and Santos, Ricardo
- Abstract
Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world’s countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome.
- Published
- 2019
12. Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants
- Author
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Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Makarova, Kira S., Wolf, Yuri I., Iranzo, Jaime, Shmakov, Sergey A., Alkhnbashi, Omer S., Brouns, Stan J.J., Charpentier, Emmanuelle, Cheng, David, Haft, Daniel H., Horvath, Philippe, Moineau, Sylvain, Mojica, Francisco J.M., Scott, David, Shah, Shiraz A., Siksnys, Virginijus, Terns, Michael P., Venclovas, Česlovas, White, Malcolm F., Yakunin, Alexander F., Yan, Winston, Zhang, Feng, Garrett, Roger A., Backofen, Rolf, Oost, John van der, Barrangou, Rodolphe, Koonin, Eugene V., Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Makarova, Kira S., Wolf, Yuri I., Iranzo, Jaime, Shmakov, Sergey A., Alkhnbashi, Omer S., Brouns, Stan J.J., Charpentier, Emmanuelle, Cheng, David, Haft, Daniel H., Horvath, Philippe, Moineau, Sylvain, Mojica, Francisco J.M., Scott, David, Shah, Shiraz A., Siksnys, Virginijus, Terns, Michael P., Venclovas, Česlovas, White, Malcolm F., Yakunin, Alexander F., Yan, Winston, Zhang, Feng, Garrett, Roger A., Backofen, Rolf, Oost, John van der, Barrangou, Rodolphe, and Koonin, Eugene V.
- Abstract
The number and diversity of known CRISPR–Cas systems have substantially increased in recent years. Here, we provide an updated evolutionary classification of CRISPR–Cas systems and cas genes, with an emphasis on the major developments that have occurred since the publication of the latest classification, in 2015. The new classification includes 2 classes, 6 types and 33 subtypes, compared with 5 types and 16 subtypes in 2015. A key development is the ongoing discovery of multiple, novel class 2 CRISPR–Cas systems, which now include 3 types and 17 subtypes. A second major novelty is the discovery of numerous derived CRISPR–Cas variants, often associated with mobile genetic elements that lack the nucleases required for interference. Some of these variants are involved in RNA-guided transposition, whereas others are predicted to perform functions distinct from adaptive immunity that remain to be characterized experimentally. The third highlight is the discovery of numerous families of ancillary CRISPR-linked genes, often implicated in signal transduction. Together, these findings substantially clarify the functional diversity and evolutionary history of CRISPR–Cas.
- Published
- 2019
13. Global phylogeography and ancient evolution of the widespread human gut virus crAssphage
- Author
-
Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Edwards, Robert A., Vega, Alejandro A., Norman, Holly M., Ohaeri, Maria, Levi, Kyle, Dinsdale, Elizabeth A., Cinek, Ondrej, Aziz, Ramy K., McNair, Katelyn, Barr, Jeremy J., Bibby, Kyle, Shimashita, John, Stachler, Elyse N., Stene, Lars C., Strain, Ronan, Stumpf, Rebecca, Torres, Pedro J., Twaddle, Alan, Ugochi Ibekwe, MaryAnn, Villagra, Nicolás, Wandro, Stephen, Brouns, Stan J.J., White, Bryan, Whiteley, Andy, Whiteson, Katrine L., Wijmenga, Cisca, Zambrano, Maria M., Zschach, Henrike, Dutilh, Bas E., Cazares, Adrian, Jonge, Patrick A. de, Desnues, Christelle, Díaz Muñoz, Samuel L., Fineran, Peter C., Kurilshikov, Alexander, Lavigne, Rob, Mazankova, Karla, McCarthy, David T., Nobrega, Franklin L., Reyes Muñoz, Alejandro, Tapia, German, Trefault, Nicole, Tyakht, Alexander V., Vinuesa, Pablo, Wagemans, Jeroen, Zhernakova, Alexandra, Aarestrup, Frank M., Ahmadov, Gunduz, Alassaf, Abeer, Anton, Josefa, Asangba, Abigail, Billings, Emma K., Cantu, Vito Adrian, Carlton, Jane M., Cazares, Daniel, Cho, Gyu-Sung, Condeff, Tess, Cortés, Pilar, Cranfield, Mike, Cuevas, Daniel A., De la Iglesia, Rodrigo, Decewicz, Przemyslaw, Doane, Michael P., Dominy, Nathaniel J., Dziewit, Lukasz, Elwasila, Bashir Mukhtar, Eren, A. Murat, Franz, Charles, Fu, Jingyuan, Garcia-Aljaro, Cristina, Ghedin, Elodie, Gulino, Kristen M., Haggerty, John M., Head, Steven R., Hendriksen, Rene S., Hill, Colin, Hyöty, Heikki, Ilina, Elena N., Irwin, Mitchell T., Jeffries, Thomas C., Jofre, Juan, Junge, Randall E., Kelley, Scott T., Mirzaei, Mohammadali Khan, Kowalewski, Martin, Kumaresan, Deepak, Leigh, Steven R., Lipson, David, Lisitsyna, Eugenia S., Llagostera, Montserrat, Maritz, Julia M., Marr, Linsey C., McCann, Angela, Molshanski-Mor, Shahar, Monteiro, Silvia, Moreira-Grez, Benjamin, Morris, Megan, Mugisha, Lawrence, Muniesa, Maite, Neve, Horst, Nguyen, Nam-phuong, Nigro, Olivia D., Nilsson, Anders S., O’Connell, Taylor, Odeh, Rasha, Oliver, Andrew, Piuri, Mariana, Prussin II, Aaron J., Qimron, Udi, Quan, Zhe-Xue, Rainetova, Petra, Ramírez-Rojas, Adán, Raya, Raul, Reasor, Kim, Rice, Gillian A.O., Rossi, Alessandro, Santos, Ricardo, Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Edwards, Robert A., Vega, Alejandro A., Norman, Holly M., Ohaeri, Maria, Levi, Kyle, Dinsdale, Elizabeth A., Cinek, Ondrej, Aziz, Ramy K., McNair, Katelyn, Barr, Jeremy J., Bibby, Kyle, Shimashita, John, Stachler, Elyse N., Stene, Lars C., Strain, Ronan, Stumpf, Rebecca, Torres, Pedro J., Twaddle, Alan, Ugochi Ibekwe, MaryAnn, Villagra, Nicolás, Wandro, Stephen, Brouns, Stan J.J., White, Bryan, Whiteley, Andy, Whiteson, Katrine L., Wijmenga, Cisca, Zambrano, Maria M., Zschach, Henrike, Dutilh, Bas E., Cazares, Adrian, Jonge, Patrick A. de, Desnues, Christelle, Díaz Muñoz, Samuel L., Fineran, Peter C., Kurilshikov, Alexander, Lavigne, Rob, Mazankova, Karla, McCarthy, David T., Nobrega, Franklin L., Reyes Muñoz, Alejandro, Tapia, German, Trefault, Nicole, Tyakht, Alexander V., Vinuesa, Pablo, Wagemans, Jeroen, Zhernakova, Alexandra, Aarestrup, Frank M., Ahmadov, Gunduz, Alassaf, Abeer, Anton, Josefa, Asangba, Abigail, Billings, Emma K., Cantu, Vito Adrian, Carlton, Jane M., Cazares, Daniel, Cho, Gyu-Sung, Condeff, Tess, Cortés, Pilar, Cranfield, Mike, Cuevas, Daniel A., De la Iglesia, Rodrigo, Decewicz, Przemyslaw, Doane, Michael P., Dominy, Nathaniel J., Dziewit, Lukasz, Elwasila, Bashir Mukhtar, Eren, A. Murat, Franz, Charles, Fu, Jingyuan, Garcia-Aljaro, Cristina, Ghedin, Elodie, Gulino, Kristen M., Haggerty, John M., Head, Steven R., Hendriksen, Rene S., Hill, Colin, Hyöty, Heikki, Ilina, Elena N., Irwin, Mitchell T., Jeffries, Thomas C., Jofre, Juan, Junge, Randall E., Kelley, Scott T., Mirzaei, Mohammadali Khan, Kowalewski, Martin, Kumaresan, Deepak, Leigh, Steven R., Lipson, David, Lisitsyna, Eugenia S., Llagostera, Montserrat, Maritz, Julia M., Marr, Linsey C., McCann, Angela, Molshanski-Mor, Shahar, Monteiro, Silvia, Moreira-Grez, Benjamin, Morris, Megan, Mugisha, Lawrence, Muniesa, Maite, Neve, Horst, Nguyen, Nam-phuong, Nigro, Olivia D., Nilsson, Anders S., O’Connell, Taylor, Odeh, Rasha, Oliver, Andrew, Piuri, Mariana, Prussin II, Aaron J., Qimron, Udi, Quan, Zhe-Xue, Rainetova, Petra, Ramírez-Rojas, Adán, Raya, Raul, Reasor, Kim, Rice, Gillian A.O., Rossi, Alessandro, and Santos, Ricardo
- Abstract
Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world’s countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome.
- Published
- 2019
14. Global phylogeography and ancient evolution of the widespread human gut virus crAssphage
- Author
-
Edwards, Robert A., Vega, Alejandro A., Norman, Holly M., Ohaeri, Maria, Levi, Kyle, Dinsdale, Elizabeth A., Cinek, Ondrej, Aziz, Ramy K., McNair, Katelyn, Barr, Jeremy J., Bibby, Kyle, Brouns, Stan J.J., Cazares, Adrian, de Jonge, Patrick A., Desnues, Christelle, Díaz Muñoz, Samuel L., Fineran, Peter C., Kurilshikov, Alexander, Lavigne, Rob, Mazankova, Karla, McCarthy, David T., Nobrega, Franklin L., Reyes Muñoz, Alejandro, Tapia, German, Trefault, Nicole, Tyakht, Alexander V., Vinuesa, Pablo, Wagemans, Jeroen, Zhernakova, Alexandra, Aarestrup, Frank M., Ahmadov, Gunduz, Alassaf, Abeer, Anton, Josefa, Asangba, Abigail, Billings, Emma K., Cantu, Vito Adrian, Carlton, Jane M., Cazares, Daniel, Cho, Gyu-Sung, Condeff, Tess, Cortés, Pilar, Cranfield, Mike, Cuevas, Daniel A., De la Iglesia, Rodrigo, Decewicz, Przemyslaw, Doane, Michael P., Dominy, Nathaniel J., Dziewit, Lukasz, Elwasila, Bashir Mukhtar, Eren, A. Murat, Franz, Charles, Fu, Jingyuan, Garcia-Aljaro, Cristina, Ghedin, Elodie, Gulino, Kristen M., Haggerty, John M., Head, Steven R., Hendriksen, Rene S., Hill, Colin, Hyöty, Heikki, Ilina, Elena N., Irwin, Mitchell T., Jeffries, Thomas C., Jofre, Juan, Junge, Randall E., Kelley, Scott T., Khan Mirzaei, Mohammadali, Kowalewski, Martin, Kumaresan, Deepak, Leigh, Steven R., Lipson, David, Lisitsyna, Eugenia S., Llagostera, Montserrat, Maritz, Julia M., Marr, Linsey C., McCann, Angela, Molshanski-Mor, Shahar, Monteiro, Silvia, Moreira-Grez, Benjamin, Morris, Megan, Mugisha, Lawrence, Muniesa, Maite, Neve, Horst, Nguyen, Nam phuong, Nigro, Olivia D., Nilsson, Anders S., O’Connell, Taylor, Odeh, Rasha, Oliver, Andrew, Piuri, Mariana, Prussin, Aaron J., Qimron, Udi, Quan, Zhe Xue, Rainetova, Petra, Ramírez-Rojas, Adán, Raya, Raul, Reasor, Kim, Rice, Gillian A.O., Rossi, Alessandro, Santos, Ricardo, Shimashita, John, Stachler, Elyse N., Stene, Lars C., Strain, Ronan, Stumpf, Rebecca, Torres, Pedro J., Twaddle, Alan, Ugochi Ibekwe, Mary Ann, Villagra, Nicolás, Wandro, Stephen, White, Bryan, Whiteley, Andy, Whiteson, Katrine L., Wijmenga, Cisca, Zambrano, Maria M., Zschach, Henrike, Dutilh, Bas E., Edwards, Robert A., Vega, Alejandro A., Norman, Holly M., Ohaeri, Maria, Levi, Kyle, Dinsdale, Elizabeth A., Cinek, Ondrej, Aziz, Ramy K., McNair, Katelyn, Barr, Jeremy J., Bibby, Kyle, Brouns, Stan J.J., Cazares, Adrian, de Jonge, Patrick A., Desnues, Christelle, Díaz Muñoz, Samuel L., Fineran, Peter C., Kurilshikov, Alexander, Lavigne, Rob, Mazankova, Karla, McCarthy, David T., Nobrega, Franklin L., Reyes Muñoz, Alejandro, Tapia, German, Trefault, Nicole, Tyakht, Alexander V., Vinuesa, Pablo, Wagemans, Jeroen, Zhernakova, Alexandra, Aarestrup, Frank M., Ahmadov, Gunduz, Alassaf, Abeer, Anton, Josefa, Asangba, Abigail, Billings, Emma K., Cantu, Vito Adrian, Carlton, Jane M., Cazares, Daniel, Cho, Gyu-Sung, Condeff, Tess, Cortés, Pilar, Cranfield, Mike, Cuevas, Daniel A., De la Iglesia, Rodrigo, Decewicz, Przemyslaw, Doane, Michael P., Dominy, Nathaniel J., Dziewit, Lukasz, Elwasila, Bashir Mukhtar, Eren, A. Murat, Franz, Charles, Fu, Jingyuan, Garcia-Aljaro, Cristina, Ghedin, Elodie, Gulino, Kristen M., Haggerty, John M., Head, Steven R., Hendriksen, Rene S., Hill, Colin, Hyöty, Heikki, Ilina, Elena N., Irwin, Mitchell T., Jeffries, Thomas C., Jofre, Juan, Junge, Randall E., Kelley, Scott T., Khan Mirzaei, Mohammadali, Kowalewski, Martin, Kumaresan, Deepak, Leigh, Steven R., Lipson, David, Lisitsyna, Eugenia S., Llagostera, Montserrat, Maritz, Julia M., Marr, Linsey C., McCann, Angela, Molshanski-Mor, Shahar, Monteiro, Silvia, Moreira-Grez, Benjamin, Morris, Megan, Mugisha, Lawrence, Muniesa, Maite, Neve, Horst, Nguyen, Nam phuong, Nigro, Olivia D., Nilsson, Anders S., O’Connell, Taylor, Odeh, Rasha, Oliver, Andrew, Piuri, Mariana, Prussin, Aaron J., Qimron, Udi, Quan, Zhe Xue, Rainetova, Petra, Ramírez-Rojas, Adán, Raya, Raul, Reasor, Kim, Rice, Gillian A.O., Rossi, Alessandro, Santos, Ricardo, Shimashita, John, Stachler, Elyse N., Stene, Lars C., Strain, Ronan, Stumpf, Rebecca, Torres, Pedro J., Twaddle, Alan, Ugochi Ibekwe, Mary Ann, Villagra, Nicolás, Wandro, Stephen, White, Bryan, Whiteley, Andy, Whiteson, Katrine L., Wijmenga, Cisca, Zambrano, Maria M., Zschach, Henrike, and Dutilh, Bas E.
- Abstract
Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world’s countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome.
- Published
- 2019
15. Global phylogeography and ancient evolution of the widespread human gut virus crAssphage
- Author
-
Edwards, Robert A., Vega, Alejandro A., Norman, Holly M., Ohaeri, Maria, Levi, Kyle, Dinsdale, Elizabeth A., Cinek, Ondrej, Aziz, Ramy K., McNair, Katelyn, Barr, Jeremy J., Bibby, Kyle, Brouns, Stan J.J., Cazares, Adrian, de Jonge, Patrick A., Desnues, Christelle, Díaz Muñoz, Samuel L., Fineran, Peter C., Kurilshikov, Alexander, Lavigne, Rob, Mazankova, Karla, McCarthy, David T., Nobrega, Franklin L., Reyes Muñoz, Alejandro, Tapia, German, Trefault, Nicole, Tyakht, Alexander V., Vinuesa, Pablo, Wagemans, Jeroen, Zhernakova, Alexandra, Møller Aarestrup, Frank, Ahmadov, Gunduz, Alassaf, Abeer, Anton, Josefa, Asangba, Abigail, Billings, Emma K., Cantu, Vito Adrian, Carlton, Jane M., Cazares, Daniel, Cho, Gyu Sung, Condeff, Tess, Cortés, Pilar, Cranfield, Mike, Cuevas, Daniel A., De la Iglesia, Rodrigo, Decewicz, Przemyslaw, Doane, Michael P., Dominy, Nathaniel J., Dziewit, Lukasz, Elwasila, Bashir Mukhtar, Eren, A. Murat, Franz, Charles, Fu, Jingyuan, Garcia-Aljaro, Cristina, Ghedin, Elodie, Gulino, Kristen M., Haggerty, John M., Head, Steven R., Hendriksen, Rene S., Hill, Colin, Hyöty, Heikki, Ilina, Elena N., Irwin, Mitchell T., Jeffries, Thomas C., Jofre, Juan, Junge, Randall E., Kelley, Scott T., Khan Mirzaei, Mohammadali, Kowalewski, Martin, Kumaresan, Deepak, Leigh, Steven R., Lipson, David, Lisitsyna, Eugenia S., Llagostera, Montserrat, Maritz, Julia M., Marr, Linsey C., McCann, Angela, Molshanski-Mor, Shahar, Monteiro, Silvia, Moreira-Grez, Benjamin, Morris, Megan, Mugisha, Lawrence, Muniesa, Maite, Neve, Horst, Nguyen, Nam phuong, Nigro, Olivia D., Nilsson, Anders S., O’Connell, Taylor, Odeh, Rasha, Oliver, Andrew, Piuri, Mariana, Prussin, Aaron J., Qimron, Udi, Quan, Zhe Xue, Rainetova, Petra, Ramírez-Rojas, Adán, Raya, Raul, Reasor, Kim, Rice, Gillian A.O., Rossi, Alessandro, Santos, Ricardo, Shimashita, John, Stachler, Elyse N., Stene, Lars C., Strain, Ronan, Stumpf, Rebecca, Torres, Pedro J., Twaddle, Alan, Ugochi Ibekwe, Mary Ann, Villagra, Nicolás, Wandro, Stephen, White, Bryan, Whiteley, Andy, Whiteson, Katrine L., Wijmenga, Cisca, Zambrano, Maria M., Zschach, Henrike, Dutilh, Bas E., Edwards, Robert A., Vega, Alejandro A., Norman, Holly M., Ohaeri, Maria, Levi, Kyle, Dinsdale, Elizabeth A., Cinek, Ondrej, Aziz, Ramy K., McNair, Katelyn, Barr, Jeremy J., Bibby, Kyle, Brouns, Stan J.J., Cazares, Adrian, de Jonge, Patrick A., Desnues, Christelle, Díaz Muñoz, Samuel L., Fineran, Peter C., Kurilshikov, Alexander, Lavigne, Rob, Mazankova, Karla, McCarthy, David T., Nobrega, Franklin L., Reyes Muñoz, Alejandro, Tapia, German, Trefault, Nicole, Tyakht, Alexander V., Vinuesa, Pablo, Wagemans, Jeroen, Zhernakova, Alexandra, Møller Aarestrup, Frank, Ahmadov, Gunduz, Alassaf, Abeer, Anton, Josefa, Asangba, Abigail, Billings, Emma K., Cantu, Vito Adrian, Carlton, Jane M., Cazares, Daniel, Cho, Gyu Sung, Condeff, Tess, Cortés, Pilar, Cranfield, Mike, Cuevas, Daniel A., De la Iglesia, Rodrigo, Decewicz, Przemyslaw, Doane, Michael P., Dominy, Nathaniel J., Dziewit, Lukasz, Elwasila, Bashir Mukhtar, Eren, A. Murat, Franz, Charles, Fu, Jingyuan, Garcia-Aljaro, Cristina, Ghedin, Elodie, Gulino, Kristen M., Haggerty, John M., Head, Steven R., Hendriksen, Rene S., Hill, Colin, Hyöty, Heikki, Ilina, Elena N., Irwin, Mitchell T., Jeffries, Thomas C., Jofre, Juan, Junge, Randall E., Kelley, Scott T., Khan Mirzaei, Mohammadali, Kowalewski, Martin, Kumaresan, Deepak, Leigh, Steven R., Lipson, David, Lisitsyna, Eugenia S., Llagostera, Montserrat, Maritz, Julia M., Marr, Linsey C., McCann, Angela, Molshanski-Mor, Shahar, Monteiro, Silvia, Moreira-Grez, Benjamin, Morris, Megan, Mugisha, Lawrence, Muniesa, Maite, Neve, Horst, Nguyen, Nam phuong, Nigro, Olivia D., Nilsson, Anders S., O’Connell, Taylor, Odeh, Rasha, Oliver, Andrew, Piuri, Mariana, Prussin, Aaron J., Qimron, Udi, Quan, Zhe Xue, Rainetova, Petra, Ramírez-Rojas, Adán, Raya, Raul, Reasor, Kim, Rice, Gillian A.O., Rossi, Alessandro, Santos, Ricardo, Shimashita, John, Stachler, Elyse N., Stene, Lars C., Strain, Ronan, Stumpf, Rebecca, Torres, Pedro J., Twaddle, Alan, Ugochi Ibekwe, Mary Ann, Villagra, Nicolás, Wandro, Stephen, White, Bryan, Whiteley, Andy, Whiteson, Katrine L., Wijmenga, Cisca, Zambrano, Maria M., Zschach, Henrike, and Dutilh, Bas E.
- Abstract
Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world’s countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome.
- Published
- 2019
16. CRISPR-Cas Systems Reduced to a Minimum
- Author
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Almendros, Cristóbal, Kieper, Sebastian N., Brouns, Stan J.J., Almendros, Cristóbal, Kieper, Sebastian N., and Brouns, Stan J.J.
- Abstract
In two recent studies in Molecular Cell, Wright et al. (2019) report complete spacer integration by a Cas1 mini-integrase and Edraki et al. (2019) describe accurate genome editing by a small Cas9 ortholog with less stringent PAM requirements.
- Published
- 2019
17. Systematic analysis of Type I-E Escherichia coli CRISPR-Cas PAM sequences ability to promote interference and primed adaptation
- Author
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Musharova, Olga, Sitnik, Vasily, Vlot, Marnix, Savitskaya, Ekaterina, Datsenko, Kirill A., Krivoy, Andrey, Fedorov, Ivan, Semenova, Ekaterina, Brouns, Stan J.J., Severinov, Konstantin, Musharova, Olga, Sitnik, Vasily, Vlot, Marnix, Savitskaya, Ekaterina, Datsenko, Kirill A., Krivoy, Andrey, Fedorov, Ivan, Semenova, Ekaterina, Brouns, Stan J.J., and Severinov, Konstantin
- Abstract
CRISPR interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation» – acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause interference, those that support rapid interference and those that lead to reduced interference that occurs over extended periods of time. PAM variants unable to support interference also did not support primed adaptation; those that supported rapid interference led to no or low levels of adaptation, while those that caused attenuated levels of interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR interference. Extended over time interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition.
- Published
- 2019
18. Cas4-Cas1 fusions drive efficient PAM selection and control CRISPR adaptation
- Author
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Almendros, Cristóbal, Nobrega, Franklin L., McKenzie, Rebecca E., Brouns, Stan J.J., Almendros, Cristóbal, Nobrega, Franklin L., McKenzie, Rebecca E., and Brouns, Stan J.J.
- Abstract
Microbes have the unique ability to acquire immunological memories from mobile genetic invaders to protect themselves from predation. To confer CRISPR resistance, new spacers need to be compatible with a targeting requirement in the invader's DNA called the protospacer adjacent motif (PAM). Many CRISPR systems encode Cas4 proteins to ensure new spacers are integrated that meet this targeting prerequisite. Here we report that a gene fusion between cas4 and cas1 from the Geobacter sulfurreducens I-U CRISPR-Cas system is capable of introducing functional spacers carrying interference proficient TTN PAM sequences at much higher frequencies than unfused Cas4 adaptation modules. Mutations of Cas4-domain catalytic residues resulted in dramatically decreased naïve and primed spacer acquisition, and a loss of PAM selectivity showing that the Cas4 domain controls Cas1 activity. We propose the fusion gene evolved to drive the acquisition of only PAM-compatible spacers to optimize CRISPR interference.
- Published
- 2019
19. Addiction systems antagonize bacterial adaptive immunity
- Author
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van Sluijs, Lisa, van Houte, Stineke, van der Oost, John, Brouns, Stan J.J., Buckling, Angus, Westra, Edze R., van Sluijs, Lisa, van Houte, Stineke, van der Oost, John, Brouns, Stan J.J., Buckling, Angus, and Westra, Edze R.
- Abstract
CRISPR-Cas systems provide adaptive immunity against mobile genetic elements, but employment of this resistance mechanism is often reported with a fitness cost for the host. Whether or not CRISPR-Cas systems are important barriers for the horizontal spread of conjugative plasmids, which play a crucial role in the spread of antibiotic resistance, will depend on the fitness costs of employing CRISPR-based defences and the benefits of resisting conjugative plasmids. To estimate these costs and benefits we measured bacterial fitness associated with plasmid immunity using Escherichia coli and the conjugative plasmid pOX38-Cm. We find that CRISPR-mediated immunity fails to confer a fitness benefit in the absence of antibiotics, despite the large fitness cost associated with carrying the plasmid in this context. Similar to many other conjugative plasmids, pOX38-Cm carries a CcdAB toxin-anti-toxin (TA) addiction system. These addiction systems encode long-lived toxins and short-lived anti-toxins, resulting in toxic effects following the loss of the TA genes from the bacterial host. Our data suggest that the lack of a fitness benefit associated with CRISPR-mediated defence is due to expression of the TA system before plasmid detection and degradation. As most antibiotic resistance plasmids encode TA systems this could have important consequences for the role of CRISPR-Cas systems in limiting the spread of antibiotic resistance.
- Published
- 2019
20. Visualisation of dCas9 target search in vivo using an open-microscopy framework
- Author
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Martens, Koen J.A., van Beljouw, Sam P.B., van der Els, Simon, Vink, Jochem N.A., Baas, Sander, Vogelaar, George A., Brouns, Stan J.J., van Baarlen, Peter, Kleerebezem, Michiel, Hohlbein, Johannes, Martens, Koen J.A., van Beljouw, Sam P.B., van der Els, Simon, Vink, Jochem N.A., Baas, Sander, Vogelaar, George A., Brouns, Stan J.J., van Baarlen, Peter, Kleerebezem, Michiel, and Hohlbein, Johannes
- Abstract
CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires constant screening of the protospacer adjacent motif (PAM) and a 30 ms upper limit for screening was recently found. To further quantify the rapid switching between DNA-bound and freely-diffusing states of dCas9, we developed an open-microscopy framework, the miCube, and introduce Monte-Carlo diffusion distribution analysis (MC-DDA). Our analysis reveals that dCas9 is screening PAMs 40% of the time in Gram-positive Lactoccous lactis, averaging 17 ± 4 ms per binding event. Using heterogeneous dCas9 expression, we determine the number of cellular target-containing plasmids and derive the copy number dependent Cas9 cleavage. Furthermore, we show that dCas9 is not irreversibly bound to target sites but can still interfere with plasmid replication. Taken together, our quantitative data facilitates further optimization of the CRISPR-Cas toolbox.
- Published
- 2019
21. Conserved motifs in the CRISPR leader sequence control spacer acquisition levels in Type I-D CRISPR-Cas systems
- Author
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Kieper, Sebastian N., Almendros, Cristóbal, Brouns, Stan J.J., Kieper, Sebastian N., Almendros, Cristóbal, and Brouns, Stan J.J.
- Abstract
Integrating short DNA fragments at the correct leader-repeat junction is key to successful CRISPR-Cas memory formation. The Cas1-2 proteins are responsible to carry out this process. However, the CRISPR adaptation process additionally requires a DNA element adjacent to the CRISPR array, called leader, to facilitate efficient localization of the correct integration site. In this work, we introduced the core CRISPR adaptation genes cas1 and cas2 from the Type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and assessed spacer integration efficiency. Truncation of the leader resulted in a significant reduction of spacer acquisition levels and revealed the importance of different conserved regions for CRISPR adaptation rates. We found three conserved sequence motifs in the leader of I-D CRISPR arrays that each affected spacer acquisition rates, including an integrase anchoring site. Our findings support the model in which the leader sequence is an integral part of type I-D adaptation in Synechocystis sp. acting as a localization signal for the adaptation complex to drive CRISPR adaptation at the first repeat of the CRISPR array.
- Published
- 2019
22. Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation
- Author
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Kieper, Sebastian N., Almendros, Cristóbal, Behler, Juliane, McKenzie, Rebecca E., Nobrega, Franklin L., Haagsma, Anna C., Vink, Jochem N.A., Hess, Wolfgang R., Brouns, Stan J.J., Kieper, Sebastian N., Almendros, Cristóbal, Behler, Juliane, McKenzie, Rebecca E., Nobrega, Franklin L., Haagsma, Anna C., Vink, Jochem N.A., Hess, Wolfgang R., and Brouns, Stan J.J.
- Abstract
CRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II CRISPR-Cas systems encode Cas4 proteins for which the role is unknown. Here, we introduced the CRISPR adaptation genes cas1, cas2, and cas4 from the type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and observed that cas4 is strictly required for the selection of targets with protospacer adjacent motifs (PAMs) conferring I-D CRISPR interference in the native host Synechocystis. We propose a model in which Cas4 assists the CRISPR adaptation complex Cas1-2 by providing DNA substrates tailored for the correct PAM. Introducing functional spacers that target DNA sequences with the correct PAM is key to successful CRISPR interference, providing a better chance of surviving infection by mobile genetic elements. Kieper et al. demonstrate that the ubiquitous protein Cas4 assists Cas1 and Cas2 in the selection of new CRISPR spacers with a PAM licensing efficient CRISPR interference.
- Published
- 2018
23. Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR–Cas effector complexes
- Author
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Vlot, Marnix, Houkes, Joep, Lochs, Silke J.A., Swarts, Daan C., Zheng, Peiyuan, Kunne, Tim, Mohanraju, Prarthana, Anders, Carolin, Jinek, Martin, Van Der Oost, John, Dickman, Mark J., Brouns, Stan J.J., Vlot, Marnix, Houkes, Joep, Lochs, Silke J.A., Swarts, Daan C., Zheng, Peiyuan, Kunne, Tim, Mohanraju, Prarthana, Anders, Carolin, Jinek, Martin, Van Der Oost, John, Dickman, Mark J., and Brouns, Stan J.J.
- Abstract
Prokaryotes encode various host defense systems that provide protection against mobile genetic elements. Restriction–modification (R–M) and CRISPR–Cas systems mediate host defense by sequence specific targeting of invasive DNA. T-even bacteriophages employ covalent modifications of nucleobases to avoid binding and therefore cleavage of their DNA by restriction endonucleases. Here, we describe that DNA glucosylation of bacteriophage genomes affects interference of some but not all CRISPR–Cas systems. We show that glucosyl modification of 5-hydroxymethylated cytosines in the DNA of bacteriophage T4 interferes with type I-E and type II-A CRISPR–Cas systems by lowering the affinity of the Cascade and Cas9–crRNA complexes for their target DNA. On the contrary, the type V-A nuclease Cas12a (also known as Cpf1) is not impaired in binding and cleavage of glucosylated target DNA, likely due to a more open structural architecture of the protein. Our results suggest that CRISPR–Cas systems have contributed to the selective pressure on phages to develop more generic solutions to escape sequence specific host defense systems.
- Published
- 2018
24. Complete genome sequence of the Escherichia coli phage Ayreon
- Author
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Vlot, Marnix, Nobrega, Franklin L., Wong, Che F.A., Liu, Yue, Brouns, Stan J.J., Vlot, Marnix, Nobrega, Franklin L., Wong, Che F.A., Liu, Yue, and Brouns, Stan J.J.
- Abstract
We report the whole-genome sequence of a new Escherichia coli temperate phage, Ayreon, comprising a linear double-stranded DNA (dsDNA) genome of 44,708 bp.
- Published
- 2018
25. Repetitive DNA Reeling by the Cascade-Cas3 Complex in Nucleotide Unwinding Steps
- Author
-
Loeff, Luuk, Brouns, Stan J.J., Joo, Chirlmin, Loeff, Luuk, Brouns, Stan J.J., and Joo, Chirlmin
- Abstract
CRISPR-Cas provides RNA-guided adaptive immunity against invading genetic elements. Interference in type I systems relies on the RNA-guided Cascade complex for target DNA recognition and the Cas3 helicase/nuclease protein for target degradation. Even though the biochemistry of CRISPR interference has been largely covered, the biophysics of DNA unwinding and coupling of the helicase and nuclease domains of Cas3 remains elusive. Here, we employed single-molecule Förster resonance energy transfer (FRET) to probe the helicase activity with high spatiotemporal resolution. We show that Cas3 remains tightly associated with the target-bound Cascade complex while reeling the DNA using a spring-loaded mechanism. This spring-loaded reeling occurs in distinct bursts of 3 bp, which underlie three successive 1-nt unwinding events. Reeling is highly repetitive, allowing Cas3 to repeatedly present its inefficient nuclease domain with single-strand DNA (ssDNA) substrate. Our study reveals that the discontinuous helicase properties of Cas3 and its tight interaction with Cascade ensure controlled degradation of target DNA only. Loeff et al. report on a single-molecule fluorescence analysis of the E. coli CRISPR-Cas3 protein. The Cas3 protein uses a spring-loaded unwinding mechanism, reeling the target DNA 3 bp at a time. Facilitated by slipping, Cas3 repeatedly presents its intrinsically inefficient nuclease domain with DNA substrate, which may contribute to promoting a robust immune response.
- Published
- 2018
26. Complete genome sequences of two T4-like Escherichia coli bacteriophages
- Author
-
Costa, Ana R., Brouns, Stan J.J., Nobrega, Franklin L., Costa, Ana R., Brouns, Stan J.J., and Nobrega, Franklin L.
- Abstract
Bacteriophages and their proteins have potential applications in biotechnology for the detection and control of bacterial diseases. Here, we describe the sequencing and genome annotations of two strictly virulent Escherichia coli bacteriophages that may be explored for biocontrol strategies and to expand the understanding of phage-host interactions.
- Published
- 2018
27. Role of nucleotide identity in effective CRISPR target escape mutations
- Author
-
Künne, Tim, Zhu, Yifan, da Silva, Fausia, Konstantinides, Nico, McKenzie, Rebecca E., Jackson, Ryan N., Brouns, Stan J.J., Künne, Tim, Zhu, Yifan, da Silva, Fausia, Konstantinides, Nico, McKenzie, Rebecca E., Jackson, Ryan N., and Brouns, Stan J.J.
- Abstract
Prokaryotes use primed CRISPR adaptation to update their memory bank of spacers against invading genetic elements that have escaped CRISPR interference through mutations in their protospacer target site. We previously observed a trend that nucleotide-dependent mismatches between crRNA and the protospacer strongly influence the efficiency of primed CRISPR adaptation. Here we show that guanine-substitutions in the target strand of the protospacer are highly detrimental to CRISPR interference and interference-dependent priming, while cytosine-substitutions are more readily tolerated. Furthermore, we show that this effect is based on strongly decreased binding affinity of the effector complex Cascade for guanine-mismatched targets, while cytosine-mismatched targets only minimally affect target DNA binding. Structural modeling of Cascade-bound targets with mismatches shows that steric clashes of mismatched guanines lead to unfavorable conformations of the RNA-DNA duplex. This effect has strong implications for the natural selection of target site mutations that lead to effective escape from type I CRISPR-Cas systems.
- Published
- 2018
28. Cas3-Derived Target DNA Degradation Fragments Fuel Primed CRISPR Adaptation
- Author
-
Künne, Tim, Kieper, Sebastian N., Bannenberg, Jasper W., Vogel, Anne, Miellet, Willem R., Klein, Misha, Depken, Martin, Suarez-Diez, Maria, Brouns, Stan J.J., Künne, Tim, Kieper, Sebastian N., Bannenberg, Jasper W., Vogel, Anne, Miellet, Willem R., Klein, Misha, Depken, Martin, Suarez-Diez, Maria, and Brouns, Stan J.J.
- Abstract
Prokaryotes use a mechanism called priming to update their CRISPR immunological memory to rapidly counter revisiting, mutated viruses, and plasmids. Here we have determined how new spacers are produced and selected for integration into the CRISPR array during priming. We show that Cas3 couples CRISPR interference to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step, PAM-associated manner. The helicase-nuclease Cas3 pre-processes target DNA into fragments of about 30–100 nt enriched for thymine-stretches in their 3′ ends. The Cas1-2 complex further processes these fragments and integrates them sequence-specifically into CRISPR repeats by coupling of a 3′ cytosine of the fragment. Our results highlight that the selection of PAM-compliant spacers during priming is enhanced by the combined sequence specificities of Cas3 and the Cas1-2 complex, leading to an increased propensity of integrating functional CTT-containing spacers.
- Published
- 2016
29. Interference-driven spacer acquisition is dominant over naive and primed adaptation in a native CRISPR-Cas system
- Author
-
Staals, Raymond H.J., Jackson, Simon A., Biswas, Ambarish, Brouns, Stan J.J., Brown, Chris M., Fineran, Peter C., Staals, Raymond H.J., Jackson, Simon A., Biswas, Ambarish, Brouns, Stan J.J., Brown, Chris M., and Fineran, Peter C.
- Abstract
CRISPR-Cas systems provide bacteria with adaptive immunity against foreign nucleic acids by acquiring short, invader-derived sequences called spacers. Here, we use high-throughput sequencing to analyse millions of spacer acquisition events in wild-type populations of Pectobacterium atrosepticum. Plasmids not previously encountered, or plasmids that had escaped CRISPR-Cas targeting via point mutation, are used to provoke naive or primed spacer acquisition, respectively. The origin, location and order of spacer acquisition show that spacer selection through priming initiates near the site of CRISPR-Cas recognition (the protospacer), but on the displaced strand, and is consistent with 3′-5′ translocation of the Cas1:Cas2-3 acquisition machinery. Newly acquired spacers determine the location and strand specificity of subsequent spacers and demonstrate that interference-driven spacer acquisition ( € targeted acquisition') is a major contributor to adaptation in type I-F CRISPR-Cas systems. Finally, we show that acquisition of self-targeting spacers is occurring at a constant rate in wild-type cells and can be triggered by foreign DNA with similarity to the bacterial chromosome.
- Published
- 2016
30. An updated evolutionary classification of CRISPR–Cas systems
- Author
-
Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Makarova, Kira S., Wolf, Yuri I., Alkhnbashi, Omer S., Costa, Fabrizio, Shah, Shiraz A., Saunders, Sita J., Barrangou, Rodolphe, Brouns, Stan J.J., Charpentier, Emmanuelle, Haft, Daniel H., Horvath, Philippe, Moineau, Sylvain, Mojica, Francisco J.M., Terns, Rebecca M., Terns, Michael P., White, Malcolm F., Yakunin, Alexander F., Garrett, Roger A., Oost, John van der, Backofen, Rolf, Koonin, Eugene V., Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Makarova, Kira S., Wolf, Yuri I., Alkhnbashi, Omer S., Costa, Fabrizio, Shah, Shiraz A., Saunders, Sita J., Barrangou, Rodolphe, Brouns, Stan J.J., Charpentier, Emmanuelle, Haft, Daniel H., Horvath, Philippe, Moineau, Sylvain, Mojica, Francisco J.M., Terns, Rebecca M., Terns, Michael P., White, Malcolm F., Yakunin, Alexander F., Garrett, Roger A., Oost, John van der, Backofen, Rolf, and Koonin, Eugene V.
- Abstract
The evolution of CRISPR–cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR–cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR–Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized.
- Published
- 2015
31. An updated evolutionary classification of CRISPR–Cas systems
- Author
-
Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Makarova, Kira S., Wolf, Yuri I., Alkhnbashi, Omer S., Costa, Fabrizio, Shah, Shiraz A., Saunders, Sita J., Barrangou, Rodolphe, Brouns, Stan J.J., Charpentier, Emmanuelle, Haft, Daniel H., Horvath, Philippe, Moineau, Sylvain, Mojica, Francisco J.M., Terns, Rebecca M., Terns, Michael P., White, Malcolm F., Yakunin, Alexander F., Garrett, Roger A., Oost, John van der, Backofen, Rolf, Koonin, Eugene V., Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Makarova, Kira S., Wolf, Yuri I., Alkhnbashi, Omer S., Costa, Fabrizio, Shah, Shiraz A., Saunders, Sita J., Barrangou, Rodolphe, Brouns, Stan J.J., Charpentier, Emmanuelle, Haft, Daniel H., Horvath, Philippe, Moineau, Sylvain, Mojica, Francisco J.M., Terns, Rebecca M., Terns, Michael P., White, Malcolm F., Yakunin, Alexander F., Garrett, Roger A., Oost, John van der, Backofen, Rolf, and Koonin, Eugene V.
- Abstract
The evolution of CRISPR–cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR–cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR–Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized.
- Published
- 2015
32. An updated evolutionary classification of CRISPR-Cas systems
- Author
-
Makarova, Kira S., Wolf, Yuri I., Alkhnbashi, Omer S., Costa, Fabrizio, Shah, Shiraz Ali, Saunders, Sita J., Barrangou, Rodolphe, Brouns, Stan J.J., Charpentier, Emmanuelle, Haft, Daniel H., Horvath, Philippe, Moineau, Sylvain, Mojica, Francisco J.M., Terns, Rebecca M., Terns, Michael P., White, Malcolm F., Yakunin, Alexander F., Garrett, Roger Antony, van der Oost, John, Backofen, Rolf, Koonin, Eugene V., Makarova, Kira S., Wolf, Yuri I., Alkhnbashi, Omer S., Costa, Fabrizio, Shah, Shiraz Ali, Saunders, Sita J., Barrangou, Rodolphe, Brouns, Stan J.J., Charpentier, Emmanuelle, Haft, Daniel H., Horvath, Philippe, Moineau, Sylvain, Mojica, Francisco J.M., Terns, Rebecca M., Terns, Michael P., White, Malcolm F., Yakunin, Alexander F., Garrett, Roger Antony, van der Oost, John, Backofen, Rolf, and Koonin, Eugene V.
- Published
- 2015
33. CRISPR interference and priming varies with individual spacer sequences
- Author
-
Xue, Chaoyou, Seetharam, Arun S., Musharova, Olga, Severinov, Konstantin, Brouns, Stan J.J., Severin, Andrew J., Sashital, Dipali G., Xue, Chaoyou, Seetharam, Arun S., Musharova, Olga, Severinov, Konstantin, Brouns, Stan J.J., Severin, Andrew J., and Sashital, Dipali G.
- Abstract
CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems allow bacteria to adapt to infection by acquiring 'spacer' sequences from invader DNA into genomic CRISPR loci. Cas proteins use RNAs derived from these loci to target cognate sequences for destruction through CRISPR interference. Mutations in the protospacer adjacent motif (PAM) and seed regions block interference but promote rapid 'primed' adaptation. Here, we use multiple spacer sequences to reexamine the PAM and seed sequence requirements for interference and priming in the Escherichia coli Type I-E CRISPR-Cas system. Surprisingly, CRISPR interference is far more tolerant of mutations in the seed and the PAM than previously reported, and this mutational tolerance, as well as priming activity, is highly dependent on spacer sequence. We identify a large number of functional PAMs that can promote interference, priming or both activities, depending on the associated spacer sequence. Functional PAMs are preferentially acquired during unprimed 'naïve' adaptation, leading to a rapid priming response following infection. Our results provide numerous insights into the importance of both spacer and target sequences for interference and priming, and reveal that priming is a major pathway for adaptation during initial infection.
- Published
- 2015
34. Comparative genomic and functional analysis of 100 Lactobacillus rhamnosus strains and their comparison with strain GG
- Author
-
University of Helsinki, Faculty of Veterinary Medicine, University of Helsinki, Haartman Institute, University of Helsinki, Institute of Biotechnology, Douillard, Francois P., Ribbera, Angela, Kant, Ravi, Pietilä, Taija E., Järvinen, Hanna M., Messing, Marcel, Randazzo, Cinzia L., Paulin, Lars, Laine, Pia, Ritari, Jarmo, Caggia, Cinzia, Lähteinen, Tanja, Brouns, Stan J.J., Satokari, Reetta, von Ossowski, Ingemar, Reunanen, Justus, Palva, Airi, Vos de, Willem M., University of Helsinki, Faculty of Veterinary Medicine, University of Helsinki, Haartman Institute, University of Helsinki, Institute of Biotechnology, Douillard, Francois P., Ribbera, Angela, Kant, Ravi, Pietilä, Taija E., Järvinen, Hanna M., Messing, Marcel, Randazzo, Cinzia L., Paulin, Lars, Laine, Pia, Ritari, Jarmo, Caggia, Cinzia, Lähteinen, Tanja, Brouns, Stan J.J., Satokari, Reetta, von Ossowski, Ingemar, Reunanen, Justus, Palva, Airi, and Vos de, Willem M.
- Published
- 2013
35. Evolution and classification of the CRISPR–Cas systems
- Author
-
Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Makarova, Kira S., Haft, Daniel H., Barrangou, Rodolphe, Brouns, Stan J.J., Charpentier, Emmanuelle, Horvath, Philippe, Moineau, Sylvain, Mojica, Francisco J.M., Wolf, Yuri I., Yakunin, Alexander F., Oost, John van der, Koonin, Eugene V., Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Makarova, Kira S., Haft, Daniel H., Barrangou, Rodolphe, Brouns, Stan J.J., Charpentier, Emmanuelle, Horvath, Philippe, Moineau, Sylvain, Mojica, Francisco J.M., Wolf, Yuri I., Yakunin, Alexander F., Oost, John van der, and Koonin, Eugene V.
- Abstract
The CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR–Cas systems and Cas proteins. Three major types of CRISPR–Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR–Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a 'polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR–cas loci.
- Published
- 2011
36. Evolution and classification of the CRISPR–Cas systems
- Author
-
Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Makarova, Kira S., Haft, Daniel H., Barrangou, Rodolphe, Brouns, Stan J.J., Charpentier, Emmanuelle, Horvath, Philippe, Moineau, Sylvain, Mojica, Francisco J.M., Wolf, Yuri I., Yakunin, Alexander F., Oost, John van der, Koonin, Eugene V., Universidad de Alicante. Departamento de Fisiología, Genética y Microbiología, Makarova, Kira S., Haft, Daniel H., Barrangou, Rodolphe, Brouns, Stan J.J., Charpentier, Emmanuelle, Horvath, Philippe, Moineau, Sylvain, Mojica, Francisco J.M., Wolf, Yuri I., Yakunin, Alexander F., Oost, John van der, and Koonin, Eugene V.
- Abstract
The CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR–Cas systems and Cas proteins. Three major types of CRISPR–Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR–Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a 'polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR–cas loci.
- Published
- 2011
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