2024-03-29T11:42:37Zhttp://repository.helmholtz-hzi.de/oai/requestoai:repository.helmholtz-hzi.de:10033/6216012019-08-30T11:29:12Zcom_10033_620968col_10033_621258
The Francisella novicida Cas12a is sensitive to the structure downstream of the terminal repeat in CRISPR arrays.
Liao, Chunyu
Slotkowski, Rebecca A
Achmedov, Tatjana
Beisel, Chase L
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
CRISPR
Cpf1
RNA structure
TXTL
terminator
The Class 2 Type V-A CRISPR effector protein Cas12a/Cpf1 has gained widespread attention in part because of the ease in achieving multiplexed genome editing, gene regulation, and DNA detection. Multiplexing derives from the ability of Cas12a alone to generate multiple guide RNAs from a transcribed CRISPR array encoding alternating conserved repeats and targeting spacers. While array design has focused on how to optimize guide-RNA sequences, little attention has been paid to sequences outside of the CRISPR array. Here, we show that a structured hairpin located immediately downstream of the 3' repeat interferes with utilization of the adjacent encoded guide RNA by Francisella novicida (Fn)Cas12a. We first observed that a synthetic Rho-independent terminator immediately downstream of an array impaired DNA cleavage based on plasmid clearance in E. coli and DNA cleavage in a cell-free transcription-translation (TXTL) system. TXTL-based cleavage assays further revealed that inhibition was associated with incomplete processing of the transcribed CRISPR array and could be attributed to the stable hairpin formed by the terminator. We also found that the inhibitory effect partially extended to upstream spacers in a multi-spacer array. Finally, we found that removing the terminal repeat from the array increased the inhibitory effect, while replacing this repeat with an unprocessable terminal repeat from a native FnCas12a array restored cleavage activity directed by the adjacent encoded guide RNA. Our study thus revealed that sequences surrounding a CRISPR array can interfere with the function of a CRISPR nuclease, with implications for the design and evolution of CRISPR arrays.
2018-12-04T13:53:22Z
2018-12-04T13:53:22Z
2018-10-12
Article
1555-8584
30252595
10.1080/15476286.2018.1526537
http://hdl.handle.net/10033/621601
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
RNA biology
oai:repository.helmholtz-hzi.de:10033/6216682019-08-30T11:33:28Zcom_10033_620968col_10033_621258
Distinct timescales of RNA regulators enable the construction of a genetic pulse generator.
Westbrook, Alexandra
Tang, Xun
Marshall, Ryan
Maxwell, Colin S
Chappell, James
Agrawal, Deepak K
Dunlop, Mary J
Noireaux, Vincent
Beisel, Chase L
Lucks, Julius
Franco, Elisa
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
Bayesian methods
CRISPRi
RNA-based circuits
model-guided design
sRNA
To build complex genetic networks with predictable behaviours, synthetic biologists use libraries of modular parts that can be characterized in isolation and assembled together to create programmable higher-order functions. Characterization experiments and computational models for gene regulatory parts operating in isolation are routinely employed to predict the dynamics of interconnected parts and guide the construction of new synthetic devices. Here, we individually characterize two modes of RNA-based transcriptional regulation, using small transcription activating RNAs (STARs) and CRISPR interference (CRISPRi), and show how their distinct regulatory timescales can be used to engineer a composed feedforward loop that creates a pulse of gene expression. We use a cell-free transcription-translation system (TXTL) to rapidly characterize the system, and we apply Bayesian inference to extract kinetic parameters for an ODE-based mechanistic model. We then demonstrate in simulation and verify with TXTL experiments that the simultaneous regulation of a single gene target with STARs and CRISPRi leads to a pulse of gene expression. Our results suggest the modularity of the two regulators in an integrated genetic circuit, and we anticipate that construction and modelling frameworks that can leverage this modularity will become increasingly important as synthetic circuits increase in complexity. This article is protected by copyright. All rights reserved.
2019-01-28T14:18:34Z
2019-01-28T14:18:34Z
2019-01-13
Article
Biotechnol Bioeng. 2019 Jan 13. doi: 10.1002/bit.26918
1097-0290
30636320
10.1002/bit.26918
http://hdl.handle.net/10033/621668
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Wiley-Blackwell
Biotechnology and bioengineering
oai:repository.helmholtz-hzi.de:10033/6217502019-08-30T11:32:10Zcom_10033_620968col_10033_621258
Bacterial Adaptation to the Host's Diet Is a Key Evolutionary Force Shaping Drosophila-Lactobacillus Symbiosis.
Martino, Maria Elena
Joncour, Pauline
Leenay, Ryan
Gervais, Hugo
Shah, Malay
Hughes, Sandrine
Gillet, Benjamin
Beisel, Chase
Leulier, François
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
Drosophila
experimental evolution
lactobacilli
microbiota
symbiosis
Animal-microbe facultative symbioses play a fundamental role in ecosystem and organismal health. Yet, due to the flexible nature of their association, the selection pressures that act on animals and their facultative symbionts remain elusive. Here we apply experimental evolution to Drosophila melanogaster associated with its growth-promoting symbiont Lactobacillus plantarum, representing a well-established model of facultative symbiosis. We find that the diet of the host, rather than the host itself, is a predominant driving force in the evolution of this symbiosis. Furthermore, we identify a mechanism resulting from the bacterium's adaptation to the diet, which confers growth benefits to the colonized host. Our study reveals that bacterial adaptation to the host's diet may be the foremost step in determining the evolutionary course of a facultative animal-microbe symbiosis.
2019-04-16T09:57:57Z
2019-04-16T09:57:57Z
2018-07-11
Article
Cell Host Microbe. 2018 Jul 11;24(1):109-119.e6. doi: 10.1016/j.chom.2018.06.001 Epub 2018 Jun 28
1934-6069
30008290
10.1016/j.chom.2018.06.001
http://hdl.handle.net/10033/621750
Cell Host and Microbe
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Elsevier
Cell host & microbe
oai:repository.helmholtz-hzi.de:10033/6218582019-08-30T11:27:09Zcom_10033_620968col_10033_621258
An enhanced assay to characterize anti-CRISPR proteins using a cell-free transcription-translation system.
Wandera, Katharina G
Collins, Scott P
Wimmer, Franziska
Marshall, Ryan
Noireaux, Vincent
Beisel, Chase L
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
Anti-CRISPR proteins
Cas9
Genome editing
TXTL
sgRNA
The characterization of CRISPR-Cas immune systems in bacteria was quickly followed by the discovery of anti-CRISPR proteins (Acrs) in bacteriophages. These proteins block different steps of CRISPR-based immunity and, as some inhibit Cas nucleases, can offer tight control over CRISPR technologies. While Acrs have been identified against a few CRISPR-Cas systems, likely many more await discovery and application. Here, we report a rapid and scalable method for characterizing putative Acrs against Cas nucleases using an E. coli-derived cell-free transcription-translation system. Using known Acrs against type II Cas9 nucleases as models, we demonstrate how the method can be used to measure the inhibitory activity of individual Acrs in under two days. We also show how the method can overcome non-specific inhibition of gene expression observed for some Acrs. In total, the method should accelerate the interrogation and application of Acrs as CRISPR-Cas inhibitors.
2019-07-10T14:37:17Z
2019-07-10T14:37:17Z
2019-05-21
Article
Methods. 2019 May 21. pii: S1046-2023(19)30001-5. doi: 10.1016/j.ymeth.2019.05.014.
1095-9130
31121300
10.1016/j.ymeth.2019.05.014
http://hdl.handle.net/10033/621858
Methods
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Elsevier
Methods (San Diego, Calif.)
oai:repository.helmholtz-hzi.de:10033/6218952019-08-30T11:26:11Zcom_10033_620968col_10033_621258
Barriers to genome editing with CRISPR in bacteria.
Vento, Justin M
Crook, Nathan
Beisel, Chase L
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
Bacteria
CRISPR
Genome editing
Nuclease
Recombineering
Genome editing is essential for probing genotype-phenotype relationships and for enhancing chemical production and phenotypic robustness in industrial bacteria. Currently, the most popular tools for genome editing couple recombineering with DNA cleavage by the CRISPR nuclease Cas9 from Streptococcus pyogenes. Although successful in some model strains, CRISPR-based genome editing has been slow to extend to the multitude of industrially relevant bacteria. In this review, we analyze existing barriers to implementing CRISPR-based editing across diverse bacterial species. We first compare the efficacy of current CRISPR-based editing strategies. Next, we discuss alternatives when the S. pyogenes Cas9 does not yield colonies. Finally, we describe different ways bacteria can evade editing and how elucidating these failure modes can improve CRISPR-based genome editing across strains. Together, this review highlights existing obstacles to CRISPR-based editing in bacteria and offers guidelines to help achieve and enhance editing in a wider range of bacterial species, including non-model strains.
2019-08-12T11:33:21Z
2019-08-12T11:33:21Z
2019-06-05
Article
J Ind Microbiol Biotechnol. 2019 Jun 5. pii: 10.1007/s10295-019-02195-1. doi: 10.1007/s10295-019-02195-1.
1476-5535
31165970
10.1007/s10295-019-02195-1
http://hdl.handle.net/10033/621895
Journal of Industrial Microbiology and Biotechnology
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Springer
Journal of industrial microbiology & biotechnology
oai:repository.helmholtz-hzi.de:10033/6219092019-08-30T11:26:12Zcom_10033_620968col_10033_621258
Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis.
Liao, Chunyu
Ttofali, Fani
Slotkowski, Rebecca A
Denny, Steven R
Cecil, Taylor D
Leenay, Ryan T
Keung, Albert J
Beisel, Chase L
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
CRISPR-Cas systems inherently multiplex through CRISPR arrays—whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.
2019-08-19T13:12:35Z
2019-08-19T13:12:35Z
2019-07-03
Article
Nat Commun. 2019 Jul 3;10(1):2948. doi: 10.1038/s41467-019-10747-3.
2041-1723
31270316
10.1038/s41467-019-10747-3
http://hdl.handle.net/10033/621909
Nature Communications
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Springer-Nature
Nature communications
oai:repository.helmholtz-hzi.de:10033/6219802019-10-17T01:33:46Zcom_10033_620968col_10033_621258
CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacter jejuni Cas9.
Dugar, Gaurav
Leenay, Ryan T
Eisenbart, Sara K
Bischler, Thorsten
Aul, Belinda U
Beisel, Chase L
Sharma, Cynthia M
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
CRISPR
Campylobacter jejuni
Cas9
RIP-seq
RNA binding proteins
RNA cleavage
crRNA
genome editing
non-coding RNA
post-transcriptional regulation
Cas9 nucleases naturally utilize CRISPR RNAs (crRNAs) to silence foreign double-stranded DNA. While recent work has shown that some Cas9 nucleases can also target RNA, RNA recognition has required nuclease modifications or accessory factors. Here, we show that the Campylobacter jejuni Cas9 (CjCas9) can bind and cleave complementary endogenous mRNAs in a crRNA-dependent manner. Approximately 100 transcripts co-immunoprecipitated with CjCas9 and generally can be subdivided through their base-pairing potential to the four crRNAs. A subset of these RNAs was cleaved around or within the predicted binding site. Mutational analyses revealed that RNA binding was crRNA and tracrRNA dependent and that target RNA cleavage required the CjCas9 HNH domain. We further observed that RNA cleavage was PAM independent, improved with greater complementarity between the crRNA and the RNA target, and was programmable in vitro. These findings suggest that C. jejuni Cas9 is a promiscuous nuclease that can coordinately target both DNA and RNA.
2019-10-16T12:56:46Z
2019-10-16T12:56:46Z
2018-03-01
Article
Mol Cell. 2018 Mar 1;69(5):893-905.e7. doi: 10.1016/j.molcel.2018.01.032.
1097-4164
29499139
10.1016/j.molcel.2018.01.032
http://hdl.handle.net/10033/621980
Molecular Cell
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Elsevier/ Cel Press
Molecular cell
oai:repository.helmholtz-hzi.de:10033/6221342020-02-15T02:01:00Zcom_10033_620968col_10033_621258
An educational module to explore CRISPR technologies with a cell-free transcription-translation system
Collias, Daphne
Marshall, Ryan
Collins, Scott P.
Beisel, Chase L.
Noireaux, Vincent
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
Cas9
CRISPR
education modules
synthetic biology
TXTL
Within the last 6 years, CRISPR-Cas systems have transitioned from adaptive defense systems in bacteria and archaea to revolutionary genome-editing tools. The resulting CRISPR technologies have driven innovations for treating genetic diseases and eradicating human pests while raising societal questions about gene editing in human germline cells as well as crop plants. Bringing CRISPR into the classroom therefore offers a means to expose students to cutting edge technologies and to promote discussions about ethical questions at the intersection of science and society. However, working with these technologies in a classroom setting has been difficult because typical experiments rely on cellular systems such as bacteria or mammalian cells. We recently reported the use of an E. coli cell-free transcription-translation (TXTL) system that simplifies the demonstration and testing of CRISPR technologies with shorter experiments and limited equipment. Here, we describe three educational modules intended to expose undergraduate students to CRISPR technologies using TXTL. The three sequential modules comprise (i) designing the RNAs that guide DNA targeting, (ii) measuring DNA cleavage activity in TXTL and (iii) testing how mutations to the targeting sequence or RNA backbone impact DNA binding and cleavage. The modules include detailed protocols, questions for group discussions or individual evaluation, and lecture slides to introduce CRISPR and TXTL. We expect these modules to allow students to experience the power and promise of CRISPR technologies in the classroom and to engage with their instructor and peers about the opportunities and potential risks for society.
2020-02-14T11:44:55Z
2020-02-14T11:44:55Z
2019-05-21
Article
19397267
10.1093/synbio/ysz005
https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85065856447&origin=inward
http://hdl.handle.net/10033/622134
Synthetic Biology
2-s2.0-85065856447
SCOPUS_ID:85065856447
en
Synthetic Biology
1
4
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Oxford Academic
oai:repository.helmholtz-hzi.de:10033/6221462020-02-19T02:01:17Zcom_10033_620968col_10033_621258
Competitive exclusion is a major bioprotective mechanism of lactobacilli against fungal spoilage in fermented milk products.
Siedler, Solvej
Rau, Martin Holm
Bidstrup, Susanne
Vento, Justin M
Aunsbjerg, Stina Dissing
Bosma, Elleke F
McNair, Laura M
Beisel, Chase L
Neves, Ana Rute
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
A prominent feature of lactic acid bacteria (LAB) is their ability to inhibit growth of spoilage organisms in food, but hitherto research efforts to establish the mechanisms underlying bioactivity focused on the production of antimicrobial compounds by LAB. We show in this study, that competitive exclusion, i.e, competition for a limited resource by different organisms, is a major mechanism of fungal growth inhibition by lactobacilli in fermented dairy products. The depletion of the essential trace element manganese by two Lactobacillus species was uncovered as the main mechanism for growth inhibition of dairy spoilage yeast and molds. A manganese transporter (MntH1), representing one of the highest expressed gene products in both lactobacilli, facilitates the exhaustive manganese scavenging. Expression of the mntH1 gene was found to be strain-dependent, affected by species co-culturing and growth phase. Further, deletion of the mntH1 gene in one of the strains resulted in loss of bioactivity, proving this gene to be important for manganese depletion. The presence of a mntH gene displayed a distinct phylogenetic pattern within the Lactobacillus genus. Moreover, assaying the bioprotective ability in fermented milk of selected lactobacilli from ten major phylogenetic groups identified a correlation between the presence of mntH and bioprotective activity. Thus, manganese scavenging emerges as a common trait within the Lactobacillus genus, but differences in expression result in some strains showing more bioprotective effect than others.In summary, competitive exclusion through ion depletion is herein reported a novel mechanism in LAB to delay growth of spoilage contaminants in dairy products.IMPORTANCE In societies that have food choices, conscious consumers demand natural solutions to keep their food healthy and fresh during storage, simultaneously reducing food waste. The use of "good bacteria" to protect food against spoilage organisms has a long successful history, even though the molecular mechanisms are not fully understood. In this study, we show that depletion of free manganese is a major bioprotective mechanism of lactobacilli in dairy products. High manganese uptake and intracellular storage provides a link to the distinct non-enzymatic manganese catalyzed oxidative stress defense mechanism, previously described for certain lactobacilli. The evaluation of representative Lactobacillus species in our study identifies multiple relevant species groups for fungal growth inhibition via manganese depletion. Hence, through the natural mechanism of nutrient depletion, the use of dedicated bioprotective lactobacilli constitutes an attractive alternative to artificial preservation.
2020-02-18T12:54:54Z
2020-02-18T12:54:54Z
2020-01-31
Article
1098-5336
32005739
10.1128/AEM.02312-19
http://hdl.handle.net/10033/622146
Applied and environmental microbiology
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
American Society of Microbiology
Applied and environmental microbiology
oai:repository.helmholtz-hzi.de:10033/6221722020-03-12T03:28:38Zcom_10033_620968col_10033_621258
CRISPR-Cas Systems and the Paradox of Self-Targeting Spacers.
Wimmer, Franziska
Beisel, Chase L
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
CRISPR-Cas
anti-CRISPR proteins
autoimmunity
gene regulation
spacer acquisition
CRISPR-Cas immune systems in bacteria and archaea record prior infections as spacers within each system's CRISPR arrays. Spacers are normally derived from invasive genetic material and direct the immune system to complementary targets as part of future infections. However, not all spacers appear to be derived from foreign genetic material and instead can originate from the host genome. Their presence poses a paradox, as self-targeting spacers would be expected to induce an autoimmune response and cell death. In this review, we discuss the known frequency of self-targeting spacers in natural CRISPR-Cas systems, how these spacers can be incorporated into CRISPR arrays, and how the host can evade lethal attack. We also discuss how self-targeting spacers can become the basis for alternative functions performed by CRISPR-Cas systems that extend beyond adaptive immunity. Overall, the acquisition of genome-targeting spacers poses a substantial risk but can aid in the host's evolution and potentially lead to or support new functionalities.
2020-02-26T09:14:18Z
2020-02-26T09:14:18Z
2019-01-01
Article
Front Microbiol. 2020 Jan 22;10:3078. doi: 10.3389/fmicb.2019.03078. eCollection 2019.
1664-302X
32038537
10.3389/fmicb.2019.03078
http://hdl.handle.net/10033/622172
Frontiers in microbiology
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Frontiers
Frontiers in microbiology
oai:repository.helmholtz-hzi.de:10033/6221842020-03-11T02:09:08Zcom_10033_620968col_10033_621258
Methods for characterizing, applying, and teaching CRISPR-Cas systems.
Beisel, Chase L
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
New drugs are desperately needed to combat methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we report screening commercial kinase inhibitors for antibacterial activity and found the anticancer drug sorafenib as major hit that effec-tively kills MRSA strains. Varying the key structural features led to the identification of a potent analogue, PK150, that showed antibacterial activity against several pathogenic strains at submicromolar concentrations. Furthermore, this antibiotic eliminated challenging persisters as well as established biofilms. PK150 holds promising therapeutic potential as it did not induce in vitro resistance, and shows oral bioavailability and in vivo efficacy. Analysis of the mode of action using chemical proteomics revealed several targets, which included interference with menaquinone biosynthesis by inhibiting demethylmenaquinone methyltrans-ferase and the stimulation of protein secretion by altering the activity of signal peptidase IB. Reduced endogenous menaquinone levels along with enhanced levels of extracellular proteins of PK150-treated bacteria support this target hypothesis. The associ-ated antibiotic effects, especially the lack of resistance development, probably stem from the compound’s polypharmacology.
2020-03-02T13:12:53Z
2020-03-02T13:12:53Z
2020-01-16
Article
Methods. 2020 Jan 16. pii: S1046-2023(20)30020-7. doi: 10.1016/j.ymeth.2020.01.004.
1095-9130
31954772
10.1016/j.ymeth.2020.01.004
http://hdl.handle.net/10033/622184
Methods
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Elsevier
Methods (San Diego, Calif.)
oai:repository.helmholtz-hzi.de:10033/6222792020-06-04T02:28:57Zcom_10033_620968col_10033_621258
Tunable self-cleaving ribozymes for modulating gene expression in eukaryotic systems.
Jacobsen, Thomas
Yi, Gloria
Al Asafen, Hadel
Jermusyk, Ashley A
Beisel, Chase L
Reeves, Gregory T
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
Advancements in the field of synthetic biology have been possible due to the development of genetic tools that are able to regulate gene expression. However, the current toolbox of gene regulatory tools for eukaryotic systems have been outpaced by those developed for simple, single-celled systems. Here, we engineered a set of gene regulatory tools by combining self-cleaving ribozymes with various upstream competing sequences that were designed to disrupt ribozyme self-cleavage. As a proof-of-concept, we were able to modulate GFP expression in mammalian cells, and then showed the feasibility of these tools in Drosophila embryos. For each system, the fold-reduction of gene expression was influenced by the location of the self-cleaving ribozyme/upstream competing sequence (i.e. 5' vs. 3' untranslated region) and the competing sequence used. Together, this work provides a set of genetic tools that can be used to tune gene expression across various eukaryotic systems.
2020-06-03T09:56:11Z
2020-06-03T09:56:11Z
2020-04-30
Article
PLoS One. 2020;15(4):e0232046. Published 2020 Apr 30. doi:10.1371/journal.pone.0232046
32352996
10.1371/journal.pone.0232046
http://hdl.handle.net/10033/622279
1932-6203
PloS one
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
PLOS
15
4
e0232046
PloS one
United States
United States
oai:repository.helmholtz-hzi.de:10033/6223452020-07-10T01:32:28Zcom_10033_620968col_10033_621258
A detailed cell-free transcription-translation-based assay to decipher CRISPR protospacer-adjacent motifs.
Maxwell, Colin S
Jacobsen, Thomas
Marshall, Ryan
Noireaux, Vincent
Beisel, Chase L
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
Cas12a
Cas9
PAM
TXTL
crRNA
sgRNA
The RNA-guided nucleases derived from the CRISPR-Cas systems in bacteria and archaea have found numerous applications in biotechnology, including genome editing, imaging, and gene regulation. However, the discovery of novel Cas nucleases has outpaced their characterization and subsequent exploitation. A key step in characterizing Cas nucleases is determining which protospacer-adjacent motif (PAM) sequences they recognize. Here, we report advances to an in vitro method based on an E. coli cell-free transcription-translation system (TXTL) to rapidly elucidate PAMs recognized by Cas nucleases. The method obviates the need for cloning Cas nucleases or gRNAs, does not require the purification of protein or RNA, and can be performed in less than a day. To advance our previously published method, we incorporated an internal GFP cleavage control to assess the extent of library cleavage as well as Sanger sequencing of the cleaved library to assess PAM depletion prior to next-generation sequencing. We also detail the methods needed to construct all relevant DNA constructs, and how to troubleshoot the assay. We finally demonstrate the technique by determining PAM sequences recognized by the Neisseria meningitidis Cas9, revealing subtle sequence requirements of this highly specific PAM. The overall method offers a rapid means to identify PAMs recognized by diverse CRISPR nucleases, with the potential to greatly accelerate our ability to characterize and harness novel CRISPR nucleases across their many uses.
2020-07-09T11:02:40Z
2020-07-09T11:02:40Z
2018-02-24
Article
Other
Methods. 2018;143:48-57. doi:10.1016/j.ymeth.2018.02.016.
29486239
10.1016/j.ymeth.2018.02.016
http://hdl.handle.net/10033/622345
1095-9130
Methods (San Diego, Calif.)
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Elsevier
143
48
57
Methods (San Diego, Calif.)
United States
United States
United States
oai:repository.helmholtz-hzi.de:10033/6224592021-07-29T12:44:12Zcom_10033_620968col_10033_621258
A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9.
Collias, D
Leenay, R T
Slotkowski, R A
Zuo, Z
Collins, S P
McGirr, B A
Liu, J
Beisel, C L
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
CRISPR technologies have overwhelmingly relied on the Streptococcus pyogenes Cas9 (SpyCas9), with its consensus NGG and less preferred NAG and NGA protospacer-adjacent motifs (PAMs). Here, we report that SpyCas9 also recognizes sequences within an N(A/C/T)GG motif. These sequences were identified on the basis of preferential enrichment in a growth-based screen in Escherichia coli. DNA binding, cleavage, and editing assays in bacteria and human cells validated recognition, with activities paralleling those for NAG(A/C/T) PAMs and dependent on the first two PAM positions. Molecular-dynamics simulations and plasmid-clearance assays with mismatch-intolerant variants supported induced-fit recognition of an extended PAM by SpyCas9 rather than recognition of NGG with a bulged R-loop. Last, the editing location for SpyCas9-derived base editors could be shifted by one nucleotide by selecting between (C/T)GG and adjacent N(C/T)GG PAMs. SpyCas9 and its enhanced variants thus recognize a larger repertoire of PAMs, with implications for precise editing, off-target predictions, and CRISPR-based immunity.
2020-09-25T09:55:08Z
2020-09-25T09:55:08Z
2020-07-15
Article
Sci Adv. 2020 Jul 15;6(29):eabb4054. doi: 10.1126/sciadv.abb4054.
32832642
10.1126/sciadv.abb4054
http://hdl.handle.net/10033/622459
2375-2548
Science advances
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
AAAS
6
29
eabb4054
Science advances
United States
oai:repository.helmholtz-hzi.de:10033/6224922021-07-29T12:44:12Zcom_10033_620968col_10033_621258
Your Base Editor Might Be Flirting with Single (Stranded) DNA: Faithful On-Target CRISPR Base Editing without Promiscuous Deamination.
Collins, Scott P
Beisel, Chase L
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
Jin et al. (2020) engineered new variants of CRISPR base editors that make precise genomic edits in rice protoplasts while minimizing untargeted mutagenesis.
2020-10-01T09:39:08Z
2020-10-01T09:39:08Z
2020-09-03
Article
Other
Mol Cell. 2020 Sep 3;79(5):703-704. doi: 10.1016/j.molcel.2020.07.030.
32888434
10.1016/j.molcel.2020.07.030
http://hdl.handle.net/10033/622492
1097-4164
Molecular cell
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Elsevier
79
5
703
704
Molecular cell
United States
oai:repository.helmholtz-hzi.de:10033/6225642021-07-29T12:44:12Zcom_10033_620968col_10033_621258
Rapid Testing of CRISPR Nucleases and Guide RNAs in an Cell-Free Transcription-Translation System.
Marshall, Ryan
Beisel, Chase L
Noireaux, Vincent
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
We present a protocol to rapidly test DNA binding and cleavage activity by CRISPR nucleases using cell-free transcription-translation (TXTL). Nuclease activity is assessed by adding DNA encoding a nuclease, a guide RNA, and a targeted reporter to a TXTL reaction and by measuring the fluorescence for several h. The reactions, performed in a few microliters, allow for parallel testing of many nucleases and guide RNAs. The protocol includes representative results for (d)Cas9 from Streptococcus pyogenes targeting a GFP reporter gene. For complete information on the generation and use of this protocol, please refer to the paper by Marshall et al. (2018).
2020-11-09T14:19:21Z
2020-11-09T14:19:21Z
2020-06-03
Article
STAR Protoc. 2020 Jun 3;1(1):100003. doi: 10.1016/j.xpro.2019.100003.
33111065
10.1016/j.xpro.2019.100003
http://hdl.handle.net/10033/622564
2666-1667
STAR protocols
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
Attribution-NonCommercial-ShareAlike 4.0 International
Elsevier (CellPress)
1
1
100003
STAR protocols
United States
oai:repository.helmholtz-hzi.de:10033/6226172021-07-29T12:44:12Zcom_10033_620968col_10033_621258
Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides.
Orsi, Enrico
Mougiakos, Ioannis
Post, Wilbert
Beekwilder, Jules
Dompè, Marco
Eggink, Gerrit
van der Oost, John
Kengen, Servé W M
Weusthuis, Ruud A
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
Growth-independent production
Isoprenoid biosynthesis
MEP
MVA
PHB
Rhodobacter sphaeroides
Microbial cell factories are usually engineered and employed for cultivations that combine product synthesis with growth. Such a strategy inevitably invests part of the substrate pool towards the generation of biomass and cellular maintenance. Hence, engineering strains for the formation of a specific product under non-growth conditions would allow to reach higher product yields. In this respect, isoprenoid biosynthesis represents an extensively studied example of growth-coupled synthesis with rather unexplored potential for growth-independent production. Rhodobacter sphaeroides is a model bacterium for isoprenoid biosynthesis, either via the native 2-methyl-d-erythritol 4-phosphate (MEP) pathway or the heterologous mevalonate (MVA) pathway, and for poly-β-hydroxybutyrate (PHB) biosynthesis.
2020-11-26T15:09:52Z
2020-11-26T15:09:52Z
2020-07-13
Article
Biotechnol Biofuels. 2020 Jul 13;13:123. doi: 10.1186/s13068-020-01765-1.
1754-6834
32684976
10.1186/s13068-020-01765-1
http://hdl.handle.net/10033/622617
Biotechnology for biofuels
en
info:eu-repo/grantAgreement/EC/H2020/834279
http://creativecommons.org/licenses/by/4.0/
openAccess
Attribution 4.0 International
BMC
13
123
Biotechnology for biofuels
England
oai:repository.helmholtz-hzi.de:10033/6227212021-07-29T12:44:12Zcom_10033_620968col_10033_621258
CRISPR technologies and the search for the PAM-free nuclease.
Collias, Daphne
Beisel, Chase L
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
The ever-expanding set of CRISPR technologies and their programmable RNA-guided nucleases exhibit remarkable flexibility in DNA targeting. However, this flexibility comes with an ever-present constraint: the requirement for a protospacer adjacent motif (PAM) flanking each target. While PAMs play an essential role in self/nonself discrimination by CRISPR-Cas immune systems, this constraint has launched a far-reaching expedition for nucleases with relaxed PAM requirements. Here, we review ongoing efforts toward realizing PAM-free nucleases through natural ortholog mining and protein engineering. We also address potential consequences of fully eliminating PAM recognition and instead propose an alternative nuclease repertoire covering all possible PAM sequences.
2021-02-08T13:22:46Z
2021-02-08T13:22:46Z
2021-01-22
Review
Nat Commun. 2021 Jan 22;12(1):555. doi: 10.1038/s41467-020-20633-y.
33483498
10.1038/s41467-020-20633-y
http://hdl.handle.net/10033/622721
2041-1723
Nature communications
en
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
Nature Pulishing Group
12
1
555
Nature communications
United States
England
oai:repository.helmholtz-hzi.de:10033/6227912021-07-29T12:44:12Zcom_10033_620968col_10033_621258
Sequence-independent RNA sensing and DNA targeting by a split domain CRISPR-Cas12a gRNA switch.
Collins, Scott P
Rostain, William
Liao, Chunyu
Beisel, Chase L
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
CRISPR technologies increasingly require spatiotemporal and dosage control of nuclease activity. One promising strategy involves linking nuclease activity to a cell's transcriptional state by engineering guide RNAs (gRNAs) to function only after complexing with a 'trigger' RNA. However, standard gRNA switch designs do not allow independent selection of trigger and guide sequences, limiting gRNA switch application. Here, we demonstrate the modular design of Cas12a gRNA switches that decouples selection of these sequences. The 5' end of the Cas12a gRNA is fused to two distinct and non-overlapping domains: one base pairs with the gRNA repeat, blocking formation of a hairpin required for Cas12a recognition; the other hybridizes to the RNA trigger, stimulating refolding of the gRNA repeat and subsequent gRNA-dependent Cas12a activity. Using a cell-free transcription-translation system and Escherichia coli, we show that designed gRNA switches can respond to different triggers and target different DNA sequences. Modulating the length and composition of the sensory domain altered gRNA switch performance. Finally, gRNA switches could be designed to sense endogenous RNAs expressed only under specific growth conditions, rendering Cas12a targeting activity dependent on cellular metabolism and stress. Our design framework thus further enables tethering of CRISPR activities to cellular states.
2021-03-24T11:13:42Z
2021-03-24T11:13:42Z
2021-02-22
2021-02-22
Article
Nucleic Acids Res. 2021 Mar 18;49(5):2985-2999. doi: 10.1093/nar/gkab100.
33619539
10.1093/nar/gkab100
http://hdl.handle.net/10033/622791
1362-4962
Nucleic acids research
en
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
Oxgord Uiversity Press
49
5
2985
2999
Nucleic acids research
United States
England
oai:repository.helmholtz-hzi.de:10033/6228252021-07-29T12:44:12Zcom_10033_620968col_10033_621258
Characterization of Cas12a nucleases reveals diverse PAM profiles between closely-related orthologs.
Jacobsen, Thomas
Ttofali, Fani
Liao, Chunyu
Manchalu, Srinivas
Gray, Benjamin N
Beisel, Chase L
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
CRISPR-Cas systems comprise diverse adaptive immune systems in prokaryotes whose RNA-directed nucleases have been co-opted for various technologies. Recent efforts have focused on expanding the number of known CRISPR-Cas subtypes to identify nucleases with novel properties. However, the functional diversity of nucleases within each subtype remains poorly explored. Here, we used cell-free transcription-translation systems and human cells to characterize six Cas12a single-effector nucleases from the V-A subtype, including nucleases sharing high sequence identity. While these nucleases readily utilized each other's guide RNAs, they exhibited distinct PAM profiles and apparent targeting activities that did not track based on phylogeny. In particular, two Cas12a nucleases encoded by Prevotella ihumii (PiCas12a) and Prevotella disiens (PdCas12a) shared over 95% amino-acid identity yet recognized distinct PAM profiles, with PiCas12a but not PdCas12a accommodating multiple G's in PAM positions -2 through -4 and T in position -1. Mutational analyses transitioning PiCas12a to PdCas12a resulted in PAM profiles distinct from either nuclease, allowing more flexible editing in human cells. Cas12a nucleases therefore can exhibit widely varying properties between otherwise related orthologs, suggesting selective pressure to diversify PAM recognition and supporting expansion of the CRISPR toolbox through ortholog mining and PAM engineering.
2021-04-08T09:48:34Z
2021-04-08T09:48:34Z
2020-07-27
Article
32329776
10.1093/nar/gkaa272
http://hdl.handle.net/10033/622825
1362-4962
Nucleic acids research
en
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
48
10
5624
5638
Nucleic acids research
United States
England
oai:repository.helmholtz-hzi.de:10033/6228292021-04-17T01:35:29Zcom_10033_620968col_10033_621258
Characterization of the all-E. coli transcription-translation system myTXTL by mass spectrometry.
Garenne, David
Beisel, Chase L
Noireaux, Vincent
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
Rationale: Cell-free transcription-translation (TXTL) is becoming a popular technology to prototype and engineer biological systems outside living organisms. TXTL relies commonly on a cytoplasmic extract that provides the molecular components necessary to recapitulate gene expression in vitro, where most of the available systems are derived from E. coli. The proteinic and enzymatic composition of lysates, however, is typically unknown. In this work, we analyzed by mass spectrometry the molecular constituents of the all-E. coli TXTL platform myTXTL prepared from the E. coli strain BL21 Rosetta2.
Methods: Standard TXTL reactions were assembled and executed for 10-12 hours at 29°C. In addition to a no-DNA control, four DNA programs were executed in separate reactions to synthesize the reporter protein deGFP as well as the phages MS2, phix174 and T7. The reactions were treated according to standard procedures (trypsin treatment, cleaning) before performing liquid chromatography/mass spectrometry (LC/MS). Data analysis was performed using Sequest and protein identification using Scaffold.
Results: A total of 500-800 proteins were identified by LC/MS in the blank reactions. We organized the most abundant protein sets into several categories pertaining, in particular, to transcription, translation and ATP regeneration. The synthesis of deGFP was easily measured. The major structural proteins that compose the three phages MS2, phix174 and T7 were also identified.
Conclusions: Mass spectrometry is a practical tool to characterize biochemical solutions as complex as a cell-free TXTL reaction and to determine the presence of synthesized proteins. The data presented demonstrate that the composition of TXTL based on lysates can be used to validate some underlying molecular mechanisms implicated in cell-free protein synthesis. The composition of the lysate shows significant differences with respect to similar studies on other E. coli strains.
2021-04-16T11:45:04Z
2021-04-16T11:45:04Z
2019-05-14
Article
Rapid Commun Mass Spectrom. 2019 May 15;33(11):1036-1048. doi: 10.1002/rcm.8438.
30900355
10.1002/rcm.8438
http://hdl.handle.net/10033/622829
1097-0231
Rapid communications in mass spectrometry : RCM
en
Wiley
33
11
1036
1048
Rapid communications in mass spectrometry : RCM
England
oai:repository.helmholtz-hzi.de:10033/6229672021-08-07T03:28:31Zcom_10033_620968col_10033_621258
A genetically encoded anti-CRISPR protein constrains gene drive spread and prevents population suppression.
Taxiarchi, Chrysanthi
Beaghton, Andrea
Don, Nayomi Illansinhage
Kyrou, Kyros
Gribble, Matthew
Shittu, Dammy
Collins, Scott P
Beisel, Chase L
Galizi, Roberto
Crisanti, Andrea
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
CRISPR-based gene drives offer promising means to reduce the burden of pests and vector-borne diseases. These techniques consist of releasing genetically modified organisms carrying CRISPR-Cas nucleases designed to bias their inheritance and rapidly propagate desired modifications. Gene drives can be intended to reduce reproductive capacity of harmful insects or spread anti-pathogen effectors through wild populations, even when these confer fitness disadvantages. Technologies capable of halting the spread of gene drives may prove highly valuable in controlling, counteracting, and even reverting their effect on individual organisms as well as entire populations. Here we show engineering and testing of a genetic approach, based on the germline expression of a phage-derived anti-CRISPR protein (AcrIIA4), able to inactivate CRISPR-based gene drives and restore their inheritance to Mendelian rates in the malaria vector Anopheles gambiae. Modeling predictions and cage testing show that a single release of male mosquitoes carrying the AcrIIA4 protein can block the spread of a highly effective suppressive gene drive preventing population collapse of caged malaria mosquitoes.
2021-07-29T11:59:03Z
2021-07-29T11:59:03Z
2021-06-25
Article
Nat Commun. 2021 Jun 25;12(1):3977. doi: 10.1038/s41467-021-24214-5.
34172748
10.1038/s41467-021-24214-5
http://hdl.handle.net/10033/622967
2041-1723
Nature communications
en
http://creativecommons.org/licenses/by/4.0/
Attribution 4.0 International
Nature research
12
1
3977
Nature communications
England