@article{jacobsen_ttofali_liao_manchalu_gray_beisel_2020, title={Characterization of Cas12a nucleases reveals diverse PAM profiles between closely-related orthologs}, volume={48}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkaa272}, abstractNote={Abstract
               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.}, number={10}, journal={NUCLEIC ACIDS RESEARCH}, author={Jacobsen, Thomas and Ttofali, Fani and Liao, Chunyu and Manchalu, Srinivas and Gray, Benjamin N. and Beisel, Chase L.}, year={2020}, month={Jun}, pages={5624–5638} }
 @article{jacobsen_yi_al asafen_jermusyk_beisel_reeves_2020, title={Tunable self-cleaving ribozymes for modulating gene expression in eukaryotic systems}, volume={15}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0232046}, abstractNote={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.}, number={4}, journal={PLOS ONE}, author={Jacobsen, Thomas and Yi, Gloria and Al Asafen, Hadel and Jermusyk, Ashley A. and Beisel, Chase L. and Reeves, Gregory T.}, year={2020}, month={Apr} }
 @article{jacobsen_liao_beisel_2019, title={The Acidaminococcus sp. Cas12a nuclease recognizes GTTV and GCTV as non-canonical PAMs}, volume={366}, ISSN={["1574-6968"]}, DOI={10.1093/femsle/fnz085}, abstractNote={ABSTRACT
               The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nuclease Acidaminococcus sp. Cas12a (AsCas12a, also known as AsCpf1) has become a popular alternative to Cas9 for genome editing and other applications. AsCas12a has been associated with a TTTV protospacer-adjacent motif (PAM) as part of target recognition. Using a cell-free transcription-translation (TXTL)-based PAM screen, we discovered that AsCas12a can also recognize GTTV and, to a lesser degree, GCTV motifs. Validation experiments involving DNA cleavage in TXTL, plasmid clearance in Escherichia coli, and indel formation in mammalian cells showed that AsCas12a was able to recognize these motifs, with the GTTV motif resulting in higher cleavage efficiency compared to the GCTV motif. We also observed that the -5 position influenced the activity of DNA cleavage in TXTL and in E. coli, with a C at this position resulting in the lowest activity. Together, these results show that wild-type AsCas12a can recognize non-canonical GTTV and GCTV motifs and exemplify why the range of PAMs recognized by Cas nucleases are poorly captured with a consensus sequence.}, number={8}, journal={FEMS MICROBIOLOGY LETTERS}, author={Jacobsen, Thomas and Liao, Chunyu and Beisel, Chase L.}, year={2019}, month={Apr} }
 @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{carrell_michael d. o'connell_jacobsen_pomeroy_hayes_reeves_2017, title={A facilitated diffusion mechanism establishes the Drosophila Dorsal gradient}, volume={144}, ISSN={["1477-9129"]}, DOI={10.1242/dev.155549}, abstractNote={The transcription factor NF-κB plays an important role in the immune system, apoptosis, and inflammation. Dorsal, a Drosophila homolog of NF-κB, patterns the dorsal-ventral axis in the blastoderm embryo. During this stage, Dorsal is sequestered outside the nucleus by the IκB homolog Cactus. Toll signaling on the ventral side breaks the Dorsal/Cactus complex, allowing Dorsal to enter the nucleus to regulate target genes. Fluorescent data show that Dorsal accumulates on the ventral side of the syncytial blastoderm. Here we use both modeling and experiment to show that this accumulation is due to facilitated diffusion, or shuttling, of Dorsal/Cactus complex. We also show that active Toll receptors are limiting in wildtype embryos, which is a key factor in explaining global Dorsal gradient formation. Our results suggest that shuttling is necessary for viability of embryos from mothers with compromised dorsal levels. Therefore, Cactus not only has the primary role of regulating Dorsal nuclear import, but also a secondary role in shuttling. Given that this mechanism has been found in other, independent systems, we suggest it may be more prevalent than previously thought.}, number={23}, journal={DEVELOPMENT}, author={Carrell, Sophia N. and Michael D. O'Connell and Jacobsen, Thomas and Pomeroy, Amy E. and Hayes, Stephanie M. and Reeves, Gregory T.}, year={2017}, month={Dec}, pages={4450–4461} }