@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}, 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{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}, 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={Abstract}, 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{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{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}, 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{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={ABSTRACT}, 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} }