@article{gawlitt_collins_yu_blackman_barquist_beisel_2024, title={Expanding the flexibility of base editing for high-throughput genetic screens in bacteria}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkae174}, abstractNote={Abstract
               Genome-wide screens have become powerful tools for elucidating genotype-to-phenotype relationships in bacteria. Of the varying techniques to achieve knockout and knockdown, CRISPR base editors are emerging as promising options. However, the limited number of available, efficient target sites hampers their use for high-throughput screening. Here, we make multiple advances to enable flexible base editing as part of high-throughput genetic screening in bacteria. We first co-opt the Streptococcus canis Cas9 that exhibits more flexible protospacer-adjacent motif recognition than the traditional Streptococcus pyogenes Cas9. We then expand beyond introducing premature stop codons by mutating start codons. Next, we derive guide design rules by applying machine learning to an essentiality screen conducted in Escherichia coli. Finally, we rescue poorly edited sites by combining base editing with Cas9-induced cleavage of unedited cells, thereby enriching for intended edits. The efficiency of this dual system was validated through a conditional essentiality screen based on growth in minimal media. Overall, expanding the scope of genome-wide knockout screens with base editors could further facilitate the investigation of new gene functions and interactions in bacteria.}, journal={NUCLEIC ACIDS RESEARCH}, author={Gawlitt, Sandra and Collins, Scott P. and Yu, Yanying and Blackman, Samuel A. and Barquist, Lars and Beisel, Chase L.}, year={2024}, month={Mar} }
 @article{tanaka_collins_polkoff_fellner_2024, title={Inhibiting methanogenesis by targeting thermodynamics and enzymatic reactions in mixed cultures of rumen microbes <i>in vitro</i>}, volume={15}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2024.1322207}, abstractNote={Mitigation of enteric methane (CH 4 ) emissions from ruminant livestock represents an opportunity to improve the sustainability, productivity, and profitability of beef and dairy production. Ruminal methanogenesis can be mitigated via two primary strategies: (1) alternative electron acceptors and (2) enzymatic inhibition of methanogenic pathways. The former utilizes the thermodynamic favorability of certain reactions such as nitrate/nitrite reduction to ammonia (NH 3 ) while the latter targets specific enzymes using structural analogs of CH 4 and methanogenic cofactors such as bromochloromethane (BCM). In this study, we investigated the effects of four additives and their combinations on CH 4 production by rumen microbes in batch culture. Sodium nitrate (NaNO 3 ), sodium sulfate (Na 2 SO 4 ), and 3-nitro-1-propionate (3NPA) were included as thermodynamic inhibitors, whereas BCM was included as a enzymatic inhibitor. Individual additives were evaluated at three levels of inclusion in experiments 1 and 2. Highest level of each additive was used to determine the combined effect of NaNO 3 + Na 2 SO 4 (NS), NS + 3NPA (NSP), and NSP + BCM (NSPB) in experiments 3 and 4. Experimental diets were high, medium, and low forage diets (HF, MF, and LF, respectively) and consisted of alfalfa hay and a concentrate mix formulated to obtain the following forage to concentrate ratios: 70:30, 50:50, and 30:70, respectively. Diets with additives were placed in fermentation culture bottles and incubated in a water bath (39°C) for 6, 12, or 24h. Microbial DNA was extracted for 16S rRNA and ITS gene amplicon sequencing. In experiments 1 and 2, CH 4 concentrations in control cultures decreased in the order of LF, MF, and HF diets, whereas in experiments 3 and 4, CH 4 was highest in MF diet followed by HF and LF diets. Culture pH and NH 3 in the control decreased in the order of HF, MF, to LF as expected. NaNO 3 decreased ( p < 0.001) CH 4 and butyrate and increased acetate and propionate ( p < 0.03 and 0.003, respectively). Cultures receiving NaNO 3 had an enrichment of microorganisms capable of nitrate and nitrite reduction. 3NPA also decreased CH 4 at 6h with no further decrease at 24 h ( p < 0.001). BCM significantly inhibited methanogenesis regardless of inclusion levels as well as in the presence of the thermodynamic inhibitors ( p < 0.001) while enriching succinate producers and assimilators as well as propionate producers ( p adj < 0.05). However, individual inclusion of BCM decreased total short chain fatty acid (SCFA) concentrations ( p < 0.002). Inhibition of methanogenesis with BCM individually and in combination with the other additives increased gaseous H 2 concentrations ( p < 0.001 individually and 0.028 in combination) while decreasing acetate to propionate ratio ( p < 0.001). Only the cultures treated with BCM in combination with other additives significantly (p adj < 0.05) decreased the abundance of Methanobrevibacter expressed as log fold change. Overall, the combination of thermodynamic and enzymatic inhibitors presented a promising effect on ruminal fermentation in-vitro , inhibiting methanogenesis while optimizing the other fermentation parameters such as pH, NH 3 , and SCFAs. Here, we provide a proof of concept that the combination of an electron acceptor and a methane analog may be exploited to improve microbial efficiency via methanogenesis inhibition.}, journal={FRONTIERS IN MICROBIOLOGY}, author={Tanaka, Kairi and Collins, Scott and Polkoff, Kathryn and Fellner, Vivek}, year={2024}, month={Aug} }
 @article{wimmer_englert_wandera_alkhnbashi_collins_backofen_beisel_2023, title={Interrogating two extensively self-targeting Type I CRISPR-Cas systems in Xanthomonas albilineans reveals distinct anti-CRISPR proteins that block DNA degradation}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkad1097}, abstractNote={Abstract
               CRISPR-Cas systems store fragments of invader DNA as spacers to recognize and clear those same invaders in the future. Spacers can also be acquired from the host's genomic DNA, leading to lethal self-targeting. While self-targeting can be circumvented through different mechanisms, natural examples remain poorly explored. Here, we investigate extensive self-targeting by two CRISPR-Cas systems encoding 24 self-targeting spacers in the plant pathogen Xanthomonas albilineans. We show that the native I-C and I-F1 systems are actively expressed and that CRISPR RNAs are properly processed. When expressed in Escherichia coli, each Cascade complex binds its PAM-flanked DNA target to block transcription, while the addition of Cas3 paired with genome targeting induces cell killing. While exploring how X. albilineans survives self-targeting, we predicted putative anti-CRISPR proteins (Acrs) encoded within the bacterium's genome. Screening of identified candidates with cell-free transcription-translation systems and in E. coli revealed two Acrs, which we named AcrIC11 and AcrIF12Xal, that inhibit the activity of Cas3 but not Cascade of the respective system. While AcrF12Xal is homologous to AcrIF12, AcrIC11 shares sequence and structural homology with the anti-restriction protein KlcA. These findings help explain tolerance of self-targeting through two CRISPR-Cas systems and expand the known suite of DNA degradation-inhibiting Acrs.}, journal={NUCLEIC ACIDS RESEARCH}, author={Wimmer, Franziska and Englert, Frank and Wandera, Katharina G. and Alkhnbashi, Omer S. and Collins, Scott P. and Backofen, Rolf and Beisel, Chase L.}, year={2023}, month={Nov} }
 @article{vialetto_yu_collins_wandera_barquist_beisel_2022, title={A target expression threshold dictates invader defense and prevents autoimmunity by CRISPR-Cas13}, volume={30}, ISSN={["1934-6069"]}, DOI={10.1016/j.chom.2022.05.013}, abstractNote={CRISPR-Cas systems must enact robust immunity against foreign genetic material without inducing cytotoxic autoimmunity. For type VI systems that use Cas13 nucleases and recognize RNA targets, immune activation requires extensive CRISPR RNA (crRNA) guide-target complementarity and a target-flanking motif. Here, we report a third requirement shaping the immune response: the expression of the target transcript exceeding a threshold. We found that endogenous non-essential transcripts targeted by crRNAs rarely elicited autoimmunity. Instead, autoimmune induction required over-expressing the targeted transcripts above a threshold. A genome-wide screen confirmed target expression levels as a global determinant of cytotoxic autoimmunity and revealed that this threshold shifts with each guide-target pair. This threshold further ensured defense against a lytic bacteriophage yet allowed the tolerance of a targeted beneficial gene expressed from an invading plasmid. These findings establish target expression levels as an additional criterion for immune defense by RNA-targeting CRISPR-Cas systems, preventing autoimmunity and distinguishing pathogenic and benign invaders.}, number={8}, journal={CELL HOST & MICROBE}, author={Vialetto, Elena and Yu, Yanying and Collins, Scott P. and Wandera, Katharina G. and Barquist, Lars and Beisel, Chase L.}, year={2022}, month={Aug}, pages={1151-+} }
 @article{durmusoglu_al'abri_collins_cheng_eroglu_beisel_crook_2021, title={In Situ Biomanufacturing of Small Molecules in the Mammalian Gut by Probiotic Saccharomyces boulardii}, volume={10}, ISSN={["2161-5063"]}, url={https://doi.org/10.1021/acssynbio.0c00562}, DOI={10.1021/acssynbio.0c00562}, abstractNote={Saccharomyces boulardii is a probiotic yeast that exhibits rapid growth at 37 °C, is easy to transform, and can produce therapeutic proteins in the gut. To establish its ability to produce small molecules encoded by multigene pathways, we measured the amount and variance in protein expression enabled by promoters, terminators, selective markers, and copy number control elements. We next demonstrated efficient (>95%) CRISPR-mediated genome editing in this strain, allowing us to probe engineered gene expression across different genomic sites. We leveraged these strategies to assemble pathways enabling a wide range of vitamin precursor (β-carotene) and drug (violacein) titers. We found that S. boulardii colonizes germ-free mice stably for over 30 days and competes for niche space with commensal microbes, exhibiting short (1-2 day) gut residence times in conventional and antibiotic-treated mice. Using these tools, we enabled β-carotene synthesis (194 μg total) in the germ-free mouse gut over 14 days, estimating that the total mass of additional β-carotene recovered in feces was 56-fold higher than the β-carotene present in the initial probiotic dose. This work quantifies heterologous small molecule production titers by S. boulardii living in the mammalian gut and provides a set of tools for modulating these titers.}, number={5}, journal={ACS SYNTHETIC BIOLOGY}, publisher={American Chemical Society (ACS)}, author={Durmusoglu, Deniz and Al'Abri, Ibrahim S. and Collins, Scott P. and Cheng, Junrui and Eroglu, Abdulkerim and Beisel, Chase L. and Crook, Nathan}, year={2021}, month={May}, pages={1039–1052} }
 @article{collins_rostain_liao_beisel_2021, title={Sequence-independent RNA sensing and DNA targeting by a split domain CRISPR-Cas12a gRNA switch}, volume={49}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkab100}, abstractNote={AbstractCRISPR 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.}, number={5}, journal={NUCLEIC ACIDS RESEARCH}, author={Collins, Scott P. and Rostain, William and Liao, Chunyu and Beisel, Chase L.}, year={2021}, month={Mar}, pages={2985–2999} }
 @article{collias_leenay_slotkowski_zuo_collins_mcgirr_liu_beisel_2020, title={A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9}, volume={6}, ISBN={2375-2548}, DOI={10.1126/sciadv.abb4054}, abstractNote={SpyCas9 and its engineered variants can recognize NYGG PAMs, affecting their use for genome editing and off-target predictions.}, number={29}, journal={SCIENCE ADVANCES}, author={Collias, D. and Leenay, R. T. and Slotkowski, R. A. and Zuo, Z. and Collins, S. P. and McGirr, B. A. and Liu, J. and Beisel, C. L.}, year={2020}, month={Jul} }
 @article{collins_beisel_2020, title={Your Base Editor Might Be Flirting with Single (Stranded) DNA: Faithful On-Target CRISPR Base Editing without Promiscuous Deamination}, volume={79}, ISSN={["1097-4164"]}, DOI={10.1016/j.molcel.2020.07.030}, abstractNote={Jin et al., 2020Jin S. Fei H. Zhu Z. Luo Y. Liu J. Gao S. Zhang F. Chen Y.H. Wang Y. Gao C. Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity.Mol. Cell. 2020; 79 (this issue): 728-740Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar engineered new variants of CRISPR base editors that make precise genomic edits in rice protoplasts while minimizing untargeted mutagenesis.}, number={5}, journal={MOLECULAR CELL}, author={Collins, Scott P. and Beisel, Chase L.}, year={2020}, month={Sep}, pages={703–704} }
 @article{collias_marshall_collins_beisel_noireaux_2019, title={An educational module to explore CRISPR technologies with a cell-free transcription-translation system}, volume={4}, ISSN={["2397-7000"]}, DOI={10.1093/synbio/ysz005}, abstractNote={Abstract
               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.}, number={1}, journal={SYNTHETIC BIOLOGY}, author={Collias, Daphne and Marshall, Ryan and Collins, Scott P. and Beisel, Chase L. and Noireaux, Vincent}, year={2019} }
 @article{wandera_collins_wimmer_marshall_noireaux_beisel_2020, title={An enhanced assay to characterize anti-CRISPR proteins using a cell-free transcription-translation system}, volume={172}, ISSN={["1095-9130"]}, DOI={10.1016/j.ymeth.2019.05.014}, abstractNote={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.}, journal={METHODS}, author={Wandera, Katharina G. and Collins, Scott P. and Wimmer, Franziska and Marshall, Ryan and Noireaux, Vincent and Beisel, Chase L.}, year={2020}, month={Feb}, pages={42–50} }
 @article{marshall_maxwell_collins_jacobsen_luo_begemann_gray_january_singer_he_et al._2018, title={Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System}, volume={69}, ISSN={["1097-4164"]}, DOI={10.1016/j.molcel.2017.12.007}, abstractNote={CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression—all without protein purification or live cells. We used TXTL to measure the dynamics of DNA cleavage and gene repression for single- and multi-effector CRISPR nucleases, predict gene repression strength in E. coli, determine the specificities of 24 diverse anti-CRISPR proteins, and develop a fast and scalable screen for protospacer-adjacent motifs that was successfully applied to five uncharacterized Cpf1 nucleases. These examples underscore how TXTL can facilitate the characterization and application of CRISPR technologies across their many uses.}, number={1}, journal={MOLECULAR CELL}, author={Marshall, Ryan and Maxwell, Colin S. and Collins, Scott P. and Jacobsen, Thomas and Luo, Michelle L. and Begemann, Matthew B. and Gray, Benjamin N. and January, Emma and Singer, Anna and He, Yonghua and et al.}, year={2018}, month={Jan}, pages={146-+} }
 @article{marshall_maxwell_collins_beisel_noireaux_2017, title={Short DNA Containing chi Sites Enhances DNA Stability and Gene Expression in E-coli Cell-Free Transcription-Translation Systems}, volume={114}, ISSN={["1097-0290"]}, DOI={10.1002/bit.26333}, abstractNote={ABSTRACTEscherichia coli cell‐free transcription–translation (TXTL) systems offer versatile platforms for advanced biomanufacturing and for prototyping synthetic biological parts and devices. Production and testing could be accelerated with the use of linear DNA, which can be rapidly and cheaply synthesized. However, linear DNA is efficiently degraded in TXTL preparations from E. coli. Here, we show that double‐stranded DNA encoding χ sites—eight base‐pair sequences preferentially bound by the RecBCD recombination machinery—stabilizes linear DNA and greatly enhances the TXTL‐based expression and activity of a fluorescent reporter gene, simple regulatory cascades, and T7 bacteriophage particles. The χ‐site DNA and the DNA‐binding λ protein Gam yielded similar enhancements, and DNA with as few as four χ sites was sufficient to ensure robust gene expression in TXTL. Given the affordability and scalability of producing the short χ‐site DNA, this generalized strategy is expected to advance the broad use of TXTL systems across its many applications. Biotechnol. Bioeng. 2017;114: 2137–2141. © 2017 Wiley Periodicals, Inc.}, number={9}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Marshall, Ryan and Maxwell, Colin S. and Collins, Scott P. and Beisel, Chase L. and Noireaux, Vincent}, year={2017}, month={Sep}, pages={2137–2141} }