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Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 83,434 bioRxiv papers from 359,709 authors.

Most downloaded bioRxiv papers, all time

in category synthetic biology

765 results found. For more information, click each entry to expand.

41: Loop Assembly: a simple and open system for recursive fabrication of DNA circuits
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Posted to bioRxiv 15 Jan 2018

Loop Assembly: a simple and open system for recursive fabrication of DNA circuits
2,394 downloads synthetic biology

Bernardo Pollak, Ariel Cerda, Mihails Delmans, Simón Álamos, Tomás Moyano, Anthony West, Rodrigo A. Gutiérrez, Nicola Patron, Fernán Federici, Jim Haseloff

High efficiency methods for DNA assembly are based on sequence overlap between fragments or Type IIS restriction endonuclease cleavage and ligation. These have enabled routine assembly of synthetic DNAs of increased size and complexity. However, these techniques require customisation, elaborate vector sets and serial manipulations for the different stages of assembly. We present Loop assembly, based on a recursive approach to DNA fabrication. Alternate use of two Type IIS restriction endonucleases and corresponding vector sets allows efficient and parallel assembly of large DNA circuits. Plasmids containing standard Level 0 parts can be assembled into circuits containing 1, 4, 16 or more genes by looping between the two vector sets. The vectors also contain modular sites for hybrid assembly using sequence overlap methods. Loop assembly provides a simple generalised solution for DNA construction with standardised parts. The cloning system is provided under an OpenMTA license for unrestricted sharing and open access.

42: Expanding functional protein sequence space using generative adversarial networks
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Posted to bioRxiv 02 Oct 2019

Expanding functional protein sequence space using generative adversarial networks
2,349 downloads synthetic biology

Donatas Repecka, Vykintas Jauniskis, Laurynas Karpus, Elzbieta Rembeza, Jan Zrimec, Simona Poviloniene, Irmantas Rokaitis, Audrius Laurynenas, Wissam Abuajwa, Otto Savolainen, Rolandas Meskys, Martin KM Engqvist, Aleksej Zelezniak

De novo protein design for catalysis of any desired chemical reaction is a long standing goal in protein engineering, due to the broad spectrum of technological, scientific and medical applications. Currently, mapping protein sequence to protein function is, however, neither computationionally nor experimentally tangible [1][1],[2][2]. Here we developed ProteinGAN, a specialised variant of the generative adversarial network [3][3] that is able to ‘learn’ natural protein sequence diversity and enables the generation of functional protein sequences. ProteinGAN learns the evolutionary relationships of protein sequences directly from the complex multidimensional amino acid sequence space and creates new, highly diverse sequence variants with natural-like physical properties. Using malate dehydrogenase as a template enzyme, we show that 24% of the ProteinGAN-generated and experimentally tested sequences are soluble and display wild-type level catalytic activity in the tested conditions in vitro , even in highly mutated (>100 mutations) sequences. ProteinGAN therefore demonstrates the potential of artificial intelligence to rapidly generate highly diverse novel functional proteins within the allowed biological constraints of the sequence space. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3

43: Recombineering in Vibrio natriegens
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Posted to bioRxiv 24 Apr 2017

Recombineering in Vibrio natriegens
2,343 downloads synthetic biology

Henry H Lee, Nili Ostrov, Michaela A Gold, George M. Church

Here, we show that λ-Red homologs found in the Vibrio-associated SXT mobile element potentiate allelic exchange in V. natriegens by 10,000-fold. Specifically, we show SXT-Beta (s065), SXT-Exo (s066), and λ-Gam proteins are sufficient to enable recombination of single- and double- stranded DNA with episomal and genomic loci. We characterize and optimize episomal oligonucleotide-mediated recombineering and demonstrate recombineering at genomic loci. We further show targeted genomic deletion of the extracellular nuclease gene dns using a double-stranded DNA cassette. Continued development of this recombination technology will advance high-throughput and large-scale genetic engineering efforts to domesticate V. natriegens and to investigate its rapid growth rate.

44: Gene cloning and construction of prokaryotic and plant expression vectors of RICIN-A-Chain/PAP-S1 fusion protein and its inhibition of protein synthesis
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Posted to bioRxiv 28 Mar 2016

Gene cloning and construction of prokaryotic and plant expression vectors of RICIN-A-Chain/PAP-S1 fusion protein and its inhibition of protein synthesis
2,277 downloads synthetic biology

Yasser Hassan, Sherry L. Ogg

Pokeweed antiviral protein (PAP) is a single-chain ribosome-inactivating protein that exists in several forms isolated from various organs and at different stages of development of Phytolacca americana (pokeweed). In this study, PAP-S1, one of the two known isoforms found in seeds, was isolated and PCR amplified using primers based on the known mRNA of PAP-S2, the other known form found in seeds. The complete cDNA encoding PAP-S1 was determined here for the first time. PAP-S1 is a potent antiviral protein with many potential clinical applications. However, it was found to be dosage dependent with observed side effects at high dosage. In this study, we report the production of a recombinant antiviral peptide-fusion protein between Ricin A-chain and PAP-S1. The peptide-fusion recombinant proteins Ricin-A-Chain/PAP-S1 and PAP-S1/Ricin-A-Chain were generated by joining the N-terminus of PAP-S1 to the C-terminus of Ricin A-chain and the C-terminus of PAP-S1 to the N-terminus of Ricin A-chain respectively, and were expressed in an Escherichia coli cell free expression systems. The peptide-fusion recombinant protein Ricin-A-Chain/PAP-S1 (F2) was found to be more active than the PAP-S1/Ricin-A-chain (F1) and similar to PAP-S1 in a cell free prokaryotic environment, and both showed much stronger activity in a cell free eukaryotic environment. The DNA sequence of the complete cDNA of PAP-S1 and of the peptide-fusion protein Ricin-A-Chain/PAP-S1 with the PAP-S1 signal peptide at the N-terminus of Ricin A-chain were inserted in plant destination binary vectors for A. tumefaciens mediated transformation. It is the opinion of the authors that additional research should be done in order to determine both cytotoxicity and selectivity of fusion protein F2 compared to PAP-S1, as it could be a viable, more potent and less cytotoxic alternative to PAP-S1 alone at high dosage, for both agricultural and therapeutic applications.

45: The WPRE Improves Genetic Engineering With Site-Specific Nucleases
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Posted to bioRxiv 12 Apr 2017

The WPRE Improves Genetic Engineering With Site-Specific Nucleases
2,265 downloads synthetic biology

Jessica M. Ong, Christopher R Brown, Matthew C. Mendel, Gregory J Cost

Inclusion of the woodchuck hepatitis virus post-transcriptional response element (WPRE) in the 3′ UTR of mRNA encoding zinc-finger or TALE nucleases results in up to a fifty-fold increase in nuclease expression and a several-fold increase in nuclease-modified chromosomes. Significantly, this increase is additive with the enhancement generated by transient hypothermic shock. The WPRE-mediated improvement is seen across several types of human and mouse primary and transformed cells and is translatable in vivo to the mouse liver.

46: Complete absence of thebaine biosynthesis under home-brew fermentation conditions
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Posted to bioRxiv 13 Aug 2015

Complete absence of thebaine biosynthesis under home-brew fermentation conditions
2,243 downloads synthetic biology

Drew Endy, Stephanie Galanie, Christina D Smolke

Yeast-based biosynthesis of medicinal compounds traditionally derived from plant materials is improving. Both concerns and hopes exist for the possibility that individual small volume batch fermentations could provide distributed and independent access to a diversity of compounds some of which are now abused, illegal, or unavailable to many who need for genuine medical purposes. However, there are differences between industrial bioreactors and ‘home-brew’ fermentation. We used engineered yeast that make thebaine, a morphinan opiate, to quantify if differences in fermentation conditions impact biosynthesis yields. We used yeast that make an English ale as a positive fermentation control. We observed no production of thebaine and miniscule amounts of reticuline, an upstream biosynthetic intermediate, in home-brew fermentations; the positive control was palatable. We suggest that additional technical challenges, some of which are unknown and likely unrelated to optimized production in large-volume bioreactors, would need to be addressed for engineered yeast to ever realize home-brew biosynthesis of medicinal opiates at meaningful yields.

47: Daisy quorum drives for the genetic restoration of wild populations
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Posted to bioRxiv 10 Mar 2017

Daisy quorum drives for the genetic restoration of wild populations
2,193 downloads synthetic biology

John Min, Charleston Noble, Devora Najjar, Kevin M. Esvelt

An ideal gene drive system to alter wild populations would 1) exclusively affect organisms within the political boundaries of consenting communities, and 2) be capable of restoring any engineered population to its original genetic state. Here we describe ‘daisy quorum’ drive systems that meet these criteria by combining daisy drive with underdominance. A daisy quorum drive system is predicted to spread through a population until all of its daisy elements have been lost, at which point its fitness becomes frequency-dependent: mostly altered populations become fixed for the desired change, while engineered genes at low frequency are swiftly eliminated by natural selection. The result is an engineered population surrounded by wild-type organisms with limited mixing at the boundary. Releasing large numbers of wild-type organisms or a few bearing a population suppression element can reduce the engineered population below the quorum, triggering elimination of all engineered sequences. In principle, the technology can restore any drive-amenable population carrying introduced genes from any source to wild-type genetics. Daisy quorum systems may enable efficient, community-supported, and genetically reversible ecological engineering.

48: Selection of chromosomal DNA libraries using a multiplex CRISPR system in Saccharomyces cerevisiae
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Posted to bioRxiv 21 Jul 2014

Selection of chromosomal DNA libraries using a multiplex CRISPR system in Saccharomyces cerevisiae
2,177 downloads synthetic biology

Owen W Ryan, Jeffrey M Skerker, Matthew J. Maurer, Xin Li, Jordan C Tsai, Snigdha Poddar, Michael E Lee, Will DeLoache, John E. Dueber, Adam P. Arkin, Jamie H. D. Cate

The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over ten-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.

49: Extensive Mammalian Germline Genome Engineering
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Posted to bioRxiv 19 Dec 2019

Extensive Mammalian Germline Genome Engineering
2,174 downloads synthetic biology

Yanan Yue, Yinan Kan, Weihong Xu, Hong-Ye Zhao, Yixuan Zhou, Xiaobin Song, Jiajia Wu, Juan Xiong, Dharmendra Goswami, Meng Yang, Lydia Lamriben, Mengyuan Xu, Chi Zhang, Yu Luo, Jianxiong Guo, Shengyi Mao, Deling Jiao, Tien Dat Nguyen, Zhuo Li, Jacob V Layer, Malin Li, Violette Paragas, Michele E. Youd, Zhongquan Sun, Yuan Ding, Weilin Wang, Hongwei Dou, Lingling Song, Xueqiong Wang, Lei Le, Xin Fang, Haydy George, Ranjith Anand, Shi Yun Wang, William F. Westlin, Marc Güell, James Markmann, Wenning Qin, Yangbin Gao, Hong-jiang Wei, George M. Church, Luhan Yang

Xenotransplantation, specifically the use of porcine organs for human transplantation, has long been sought after as an alternative for patients suffering from organ failure. However, clinical application of this approach has been impeded by two main hurdles: 1) risk of transmission of porcine endogenous retroviruses (PERVs) and 2) molecular incompatibilities between donor pigs and humans which culminate in rejection of the graft. We previously demonstrated that all 25 copies of the PERV elements in the pig genome could be inactivated and live pigs successfully generated. In this study, we improved the scale of porcine germline editing from targeting a single repetitive locus with CRISPR to engineering 13 different genes using multiple genome engineering methods. we engineered the pig genome at 42 alleles using CRISPR-Cas9 and transposon and produced PERVKO·3KO·9TG pigs which carry PERV inactivation, xeno-antigen KO and 9 effective human transgenes. The engineered pigs exhibit normal physiology, fertility, and germline transmission of the edited alleles. In vitro assays demonstrated that these pigs gain significant resistance to human humoral and cell mediated damage, and coagulation dysregulations, similar to that of allotransplantation. Successful creation of PERVKO·3KO·9TG pigs represents a significant step forward towards safe and effective porcine xenotransplantation, which also represents a synthetic biology accomplishment of engineering novel functions in a living organism. One Sentence Summary Extensive genome engineering is applied to modify pigs for safe and immune compatible organs for human transplantation

50: Automated continuous evolution of proteins in vivo
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Posted to bioRxiv 22 Feb 2020

Automated continuous evolution of proteins in vivo
2,172 downloads synthetic biology

Ziwei Zhong, Brandon G Wong, Arjun Ravikumar, Garri A. Arzumanyan, Ahmad S. Khalil, Chang C. Liu

We present automated continuous evolution (ACE), a platform for the hands-free directed evolution of biomolecules. ACE pairs OrthoRep, a genetic system for continuous targeted mutagenesis of user-selected genes in vivo, with eVOLVER, a scalable and automated continuous culture device for precise, multi-parameter regulation of growth conditions. By implementing real-time feedback-controlled tuning of selection stringency with eVOLVER, genes of interest encoded on OrthoRep autonomously traversed multi-mutation adaptive pathways to reach desired functions, including drug resistance and improved enzyme activity. The durability, scalability, and speed of biomolecular evolution with ACE should be broadly applicable to protein engineering as well as prospective studies on how selection parameters and schedules shape adaptation.

51: Evaluating Protein Transfer Learning with TAPE
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Posted to bioRxiv 20 Jun 2019

Evaluating Protein Transfer Learning with TAPE
2,138 downloads synthetic biology

Roshan Rao, Nicholas Bhattacharya, Neil Thomas, Yan Duan, Xi Chen, John Canny, Pieter Abbeel, Yun S. Song

Protein modeling is an increasingly popular area of machine learning research. Semi-supervised learning has emerged as an important paradigm in protein modeling due to the high cost of acquiring supervised protein labels, but the current literature is fragmented when it comes to datasets and standardized evaluation techniques. To facilitate progress in this field, we introduce the Tasks Assessing Protein Embeddings (TAPE), a set of five biologically relevant semi-supervised learning tasks spread across different domains of protein biology. We curate tasks into specific training, validation, and test splits to ensure that each task tests biologically relevant generalization that transfers to real-life scenarios. We benchmark a range of approaches to semi-supervised protein representation learning, which span recent work as well as canonical sequence learning techniques. We find that self-supervised pretraining is helpful for almost all models on all tasks, more than doubling performance in some cases. Despite this increase, in several cases features learned by self-supervised pretraining still lag behind features extracted by state-of-the-art non-neural techniques. This gap in performance suggests a huge opportunity for innovative architecture design and improved modeling paradigms that better capture the signal in biological sequences. TAPE will help the machine learning community focus effort on scientifically relevant problems. Toward this end, all data and code used to run these experiments are available at https://github.com/songlab-cal/tape.

52: A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells
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Posted to bioRxiv 26 Dec 2018

A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells
2,110 downloads synthetic biology

João Pedro Fonseca, Alain R Bonny, G. Renuka Kumar, Andrew H Ng, Jason Town, Qiu Chang Wu, Elham Aslankoohi, Susan Y Chen, Patrick Harrigan, Lindsey C Osimiri, Amy L Kistler, Hana El-Samad

The ability to rapidly assemble and prototype cellular circuits is vital for biological research and its applications in biotechnology and medicine. Current methods that permit the assembly of DNA circuits in mammalian cells are laborious, slow, expensive and mostly not permissive of rapid prototyping of constructs. Here we present the Mammalian ToolKit (MTK), a Golden Gate-based cloning toolkit for fast, reproducible and versatile assembly of large DNA vectors and their implementation in mammalian models. The MTK consists of a curated library of characterized, modular parts that can be easily mixed and matched to combinatorially assemble one transcriptional unit with different characteristics, or a hierarchy of transcriptional units weaved into complex circuits. MTK renders many cell engineering operations facile, as showcased by our ability to use the toolkit to generate single-integration landing pads, to create and deliver libraries of protein variants and sgRNAs, and to iterate through Cas9-based prototype circuits. As a biological proof of concept, we used the MTK to successfully design and rapidly construct in mammalian cells a challenging multicistronic circuit encoding the Ebola virus (EBOV) replication complex. This construct provides a non-infectious biosafety level 2 (BSL2) cellular assay for exploring the transcription and replication steps of the EBOV viral life cycle in its host. Its construction also demonstrates how the MTK can enable important and time sensitive applications such as the rapid testing of pharmacological inhibitors of emerging BSL4 viruses that pose a major threat to human health.

53: Programmable bacteria induce durable tumor regression and systemic antitumor immunity
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Posted to bioRxiv 01 Mar 2019

Programmable bacteria induce durable tumor regression and systemic antitumor immunity
2,010 downloads synthetic biology

Sreyan Chowdhury, Taylor E. Hinchliffe, Samuel Castro, Courtney Coker, Nicholas Arpaia, Tal Danino

Synthetic biology is driving a new era of medicine through the genetic programming of living cells. This transformative approach allows for the creation of engineered systems that intelligently sense and respond to diverse environments, ultimately adding specificity and efficacy that extends beyond the capabilities of molecular-based therapeutics. One particular focus area has been the engineering of bacteria as therapeutic delivery systems to selectively release therapeutic payloads in vivo. Here, we engineered a non-pathogenic E. coli to specifically lyse within the tumor microenvironment and release an encoded nanobody antagonist of CD47 (CD47nb), an anti-phagocytic receptor commonly overexpressed in several human cancers. We show that intratumoral delivery of CD47nb by tumor-colonizing bacteria increases activation of tumor-infiltrating T cells, stimulates rapid tumor regression, prevents metastasis, and leads to long-term survival in a syngeneic tumor model. Moreover, we report that local injection of CD47nb bacteria stimulates systemic antitumor immune responses that reduce the growth of untreated tumors - providing, to the best of our knowledge, the first demonstration of an abscopal effect induced by a bacteria cancer therapy. Thus, engineered bacteria may be used for safe and local delivery of immunotherapeutic payloads leading to systemic antitumor immunity.

54: Low cost and open source multi-fluorescence imaging system for teaching and research in biology and bioengineering
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Posted to bioRxiv 27 Sep 2017

Low cost and open source multi-fluorescence imaging system for teaching and research in biology and bioengineering
1,999 downloads synthetic biology

Nuñez Isaac, Matute Tamara, Herrera Roberto, Keymer Juan, Marzullo Tim, Rudge Tim, Federici Fernán

The advent of easy-to-use open source microcontrollers, off-the-shelf electronics and customizable manufacturing technologies has facilitated the development of inexpensive scientific devices and laboratory equipment. In this study, we describe an imaging system that integrates low-cost and open-source hardware, software and genetic resources. The multi-fluorescence imaging system consists of readily available 470 nm LEDs, a Raspberry Pi camera and a set of filters made with low cost acrylics. This device allows imaging in scales ranging from single colonies to entire plates. We developed a set of genetic components (e.g. promoters, coding sequences, terminators) and vectors following the standard framework of Golden Gate, which allowed the fabrication of genetic constructs in a combinatorial, low cost and robust manner. In order to provide simultaneous imaging of multiple wavelength signals, we screened a series of long stokes shift fluorescent proteins that could be combined with cyan/green fluorescent proteins. We found CyOFP1, mBeRFP and sfGFP to be the most compatible set for 3-channel fluorescent imaging. We developed open source Python code to operate the hardware to run time-lapse experiments with automated control of illumination and camera and a Python module to analyze data and extract meaningful biological information. To demonstrate the potential application of this integral system, we tested its performance on a diverse range of imaging assays often used in disciplines such as microbial ecology, microbiology and synthetic biology. We also assessed its potential for STEM teaching in a high school environment, using it to teach biology, hardware design, optics, and programming. Together, these results demonstrate the successful integration of open source hardware, software, genetic resources and customizable manufacturing to obtain a powerful, low cost and robust system for STEM education, scientific research and bioengineering. All the resources developed here are available under open source licenses.

55: Rapid, Low-Cost Detection of Water Contaminants Using Regulated In Vitro Transcription
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Posted to bioRxiv 26 Apr 2019

Rapid, Low-Cost Detection of Water Contaminants Using Regulated In Vitro Transcription
1,951 downloads synthetic biology

Khalid K. Alam, Jaeyoung K. Jung, Matthew S. Verosloff, Phillip R. Clauer, Jeong Wook Lee, Daiana A. Capdevila, Pablo A. Pastén, David P. Giedroc, James J. Collins, Julius B. Lucks

Synthetic biology has enabled the development of powerful nucleic acid diagnostic technologies for detecting pathogens and human health biomarkers. Here we expand the reach of synthetic biology-enabled diagnostics by developing a cell-free biosensing platform that uses RNA output sensors activated by ligand induction (ROSALIND) to detect harmful contaminants in aqueous samples. ROSALIND consists of three programmable components: highly-processive RNA polymerases, allosteric transcription factors, and synthetic DNA transcription templates. Together, these components allosterically regulate the in vitro transcription of a fluorescence-activating RNA aptamer: in the absence of a target compound, transcription is blocked, while in its presence a fluorescent signal is produced. We demonstrate that ROSALIND can be configured to detect a range of water contaminants, including antibiotics, toxic small molecules, and metals. Our cell-free biosensing platform, which can be freeze-dried for field deployment, creates a new capability for point-of-use monitoring of molecular species to address growing global crises in water quality and human health.

56: Histone modifications and active gene expression are associated with enhanced CRISPR activity in de-silenced chromatin
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Posted to bioRxiv 04 Dec 2017

Histone modifications and active gene expression are associated with enhanced CRISPR activity in de-silenced chromatin
1,944 downloads synthetic biology

René Daer, Cassandra M. Barrett, Karmella A. Haynes

Recently we demonstrated that closed chromatin composed of Polycomb proteins and histone 3 lysine 27 trimethylation impedes CRISPR-mediated genome editing by blocking the accessibility of chromosomal DNA to spCas9/sgRNA. Editing efficiencies were higher in cells where the same reporter locus had not been repressed, thus we presume that silenced chromatin can be modified to generate a Cas9-accessible state. To test this idea, we exposed the locus to antagonists of Polycomb silencing: Gal4-p65, a targeted transcriptional activator, and UNC1999, a chemical inhibitor of the histone H3K27 methyltransferase EZH2. For both we observed loss of histone trimethylation. Only Gal4-p65 treatment increased target gene expression. Initial Gal4-p65 overexpression impedes Cas9 activity, while a 9-day recovery period leads to enhanced Cas9 efficiency up to 1000 bp from the Gal4 binding site. No enhancement was observed with UNC1999. These results demonstrate the strong influence of transcription-driven chromatin remodeling on CRISPR editing at closed chromatin.

57: Quantifying cellular capacity to identify gene expression designs with reduced burden
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Posted to bioRxiv 22 Dec 2014

Quantifying cellular capacity to identify gene expression designs with reduced burden
1,913 downloads synthetic biology

Francesca Ceroni, Rhys Algar, Guy-Bart Stan, Tom Ellis

Heterologous gene expression can be a significant burden to cells, consuming resources and causing decreased growth and stability. We describe here an in vivo monitor that tracks E. coli capacity changes in real-time and can be used to assay the burden synthetic constructs and their parts impose. By measuring capacity, construct designs with reduced burden can be identified and shown to predictably outperform less efficient designs, despite having equivalent expression outputs.

58: Deep Learning Of The Regulatory Grammar Of Yeast 5′ Untranslated Regions From 500,000 Random Sequences
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Posted to bioRxiv 12 May 2017

Deep Learning Of The Regulatory Grammar Of Yeast 5′ Untranslated Regions From 500,000 Random Sequences
1,912 downloads synthetic biology

Josh T. Cuperus, Benjamin Groves, Anna Kuchina, Alexander B Rosenberg, Nebojsa Jojic, Stanley Fields, Georg Seelig

Our ability to predict protein expression from DNA sequence alone remains poor, reflecting our limited understanding of cis-regulatory grammar and hampering the design of engineered genes for synthetic biology applications. Here, we generate a model that predicts the translational efficiency of the 5′ untranslated region (UTR) of mRNAs in the yeast Saccharomyces cerevisiae. We constructed a library of half a million 50-nucleotide-long random 5′ UTRs and assayed their activity in a massively parallel growth selection experiment. The resulting data allow us to quantify the impact on translation of Kozak sequence composition, upstream open reading frames (uORFs) and secondary structure. We trained a convolutional neural network (CNN) on the random library and showed that it performs well at predicting the translational efficiency of both a held-out set of the random 5′ UTRs as well as native S. cerevisiae 5′ UTRs. The model additionally was used to computationally evolve highly translating 5′ UTRs. We confirmed experimentally that the great majority of the evolved sequences lead to higher translation rates than the starting sequences, demonstrating the predictive power of this model.

59: Engineered Cpf1 Enzymes with Altered PAM Specificities
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Posted to bioRxiv 04 Dec 2016

Engineered Cpf1 Enzymes with Altered PAM Specificities
1,908 downloads synthetic biology

Linyi Gao, David B.T. Cox, Winston X Yan, John Manteiga, Martin Schneider, Takashi Yamano, Hiroshi Nishimasu, Osamu Nureki, Feng Zhang

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. Compared to other genome editing platforms, Cpf1 offers distinct advantages, such as the ability to easily target multiple genes simultaneously, as well as low rates of off-target activity. However, the Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), which has been successfully harnessed for genome editing, can only robustly cleave target sites preceded by a TTTV protospacer adjacent motif (PAM), which may limit its practical utility. To address this limitation, we used a structure- guided saturation mutagenesis screen to increase the targeting range of Cpf1. We engineered two variants of AsCpf1 with the mutations S542R/K607R and S542R/K548V/N552R that can cleave target sites with TYCV/CCCC and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity indicated that these variants retain a high level of DNA targeting specificity, which can be further improved by introducing mutations in non-PAM-interacting domains. Together, these variants increase the targeting range of AsCpf1 to one cleavage site for every ~8.7 bp in non-repetitive regions of the human genome, providing a useful addition to the CRISPR/Cas genome engineering toolbox.

60: Highly efficient CRISPR gene editing in yeast enabled by double selection
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Posted to bioRxiv 09 Feb 2018

Highly efficient CRISPR gene editing in yeast enabled by double selection
1,888 downloads synthetic biology

Philippe C Després, Alexandre K Dubé, Lou Nielly-Thibault, Nozomu Yachie, Christian R Landry

CRISPR-Cas9 loss of function (LOF) and base editing screens are powerful tools in genetics and genomics. Yeast is one of the main models in genetics and genomics, yet large-scale approaches remain to be developed in this species because of low mutagenesis rates without donor DNA. We developed a double selection strategy based on co-selection that increases LOF mutation rates, both for CRISPR-Cas9 and the Target-AID base editor. We constructed the pDYSCKO vector, which is amenable to high throughput double selection for both approaches. Using modeling, we show that this improvement provides the required increased in detection power to measure the fitness effects of thousands of mutations in typical yeast pooled screens. We also show that multiplex genome editing with Cas9 causes programmable chromosomal translocations at high frequency, suggesting that multiplex editing should be performed with caution and that base-editors could be preferable tools for LOF screens.

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