The physicist's guide to one of biotechnology's hottest new topics: CRISPR-Cas

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) constitute a multi-functional, constantly evolving immune system in bacteria and archaea cells. A heritable, molecular memory is generated of phage, plasmids, or other mobile genetic elements that attempt to attack the cell. This memory is used to recognize and interfere with subsequent invasions from the same genetic elements. This versatile prokaryotic tool has also been used to advance applications in biotechnology. Here we review a large body of CRISPR-Cas research to explore themes of evolution and selection, population dynamics, horizontal gene transfer, specific and cross-reactive interactions, cost and regulation, non-immunological CRISPR functions that boost host cell robustness, as well as applicable mechanisms for efficient and specific genetic engineering. We offer future directions that can be addressed by the physics community. Physical understanding of the CRISPR-Cas system will advance uses in biotechnology, such as developing cell lines and animal models, cell labeling and information storage, combatting antibiotic resistance, and human therapeutics.Comment: 75 pages, 15 figures, Physical Biology (2018

Engineering Open Chromatin with Synthetic Pioneer Factors: Enhancing Mammalian Transgene Expression and Improving Cas9-Mediated Genome Editing in Closed Chromatin

abstract: Chromatin is the dynamic structure of proteins and nucleic acids into which eukaryotic genomes are organized. For those looking to engineer mammalian genomes, chromatin is both an opportunity and an obstacle. While chromatin provides another tool with which to control gene expression, regional density can lead to variability in genome editing efficiency by CRISPR/Cas9 systems. Many groups have attempted to de-silence chromatin to regulate genes and enhance DNA's accessibility to nucleases, but inconsistent results leave outstanding questions. Here, I test different types of activators, to analyze changes in chromatin features that result for chromatin opening, and to identify the critical biochemical features that support artificially generated open, transcriptionally active chromatin. I designed, built, and tested a panel of synthetic pioneer factors (SPiFs) to open condensed, repressive chromatin with the aims of 1) activating repressed transgenes in mammalian cells and 2) reversing the inhibitory effects of closed chromatin on Cas9-endonuclease activity. Pioneer factors are unique in their ability to bind DNA in closed chromatin. In order to repurpose this natural function, I designed SPiFs from a Gal4 DNA binding domain, which has inherent pioneer functionality, fused with chromatin-modifying peptides with distinct functions. SPiFs with transcriptional activation as their primary mechanism were able to reverse this repression and induced a stably active state. My work also revealed the active site from proto-oncogene MYB as a novel transgene activator. To determine if MYB could be used generally to restore transgene expression, I fused it to a deactivated Cas9 and targeted a silenced transgene in native heterochromatin. The resulting activator was able to reverse silencing and can be chemically controlled with a small molecule drug. Other SPiFs in my panel did not increase gene expression. However, pretreatment with several of these expression-neutral SPiFs increased Cas9-mediated editing in closed chromatin, suggesting a crucial difference between chromatin that is accessible and that which contains genes being actively transcribed. Understanding this distinction will be vital to the engineering of stable transgenic cell lines for product production and disease modeling, as well as therapeutic applications such as restoring epigenetic order to misregulated disease cells.Dissertation/ThesisDoctoral Dissertation Biological Design 201

Synthetic Genomics

The current advances in sequencing, data mining, DNA synthesis, cloning, in silico modeling, and genome editing have opened a new field of research known as Synthetic Genomics. The main goal of this emerging area is to engineer entire synthetic genomes from scratch using pre-designed building blocks obtained by chemical synthesis and rational design. This has opened the possibility to further improve our understanding of genome fundamentals by considering the effect of the whole biological system on biological function. Moreover, the construction of non-natural biological systems has allowed us to explore novel biological functions so far not discovered in nature. This book summarizes the current state of Synthetic Genomics, providing relevant examples in this emerging field

Engineering Proteins by Domain Insertion

Protein domains are structural and functional subunits of proteins. The recombination of existing domains is a source of evolutionary innovation, as it can result in new protein features and functions. Inspired by nature, protein engineering commonly uses domain recombination in order to create artificial proteins with tailor-made properties. Customized control over protein activity, for instance, can be achieved by harnessing switchable domains and functionally linking them to effector domains. Many natural protein domains exhibit conformational changes in response to exogenous triggers. The insertion of light-switchable receptor domains into an effector protein of choice, for instance, allows the control of effector activity with light. The resulting optogenetic proteins represent powerful tools for the investigation of dynamic cellular processes with high precision in time and space. On top, optogenetic proteins enable manifold biotechnological applications and they are even considered potential candidates for future therapeutics. In this study, we first focused on CRISPR-Cas9 genome editing and applied a domain insertion strategy to genetically encoded inhibitors of the CRISPR nuclease from Neisseria meningitidis (NmeCas9), which due to its small size and high DNA sequence-specificity is of great interest for CRISPR genome editing applications. Fusing stabilizing domains to the NmeCas9 inhibitory protein AcrIIC1 allowed us to boost its inhibitory effect, thereby yielding a potent gene editing off-switch. Furthermore, the insertion of the light-responsive LOV2 domain from Avena sativa into AcrIIC3, the most potent inhibitor of NmeCas9, enabled the optogenetic control of gene editing via light-dependent NmeCas9 inhibition. Further investigation of the engineered inhibitors revealed the potential these proteins could have with respect to safe-guarding of the CRISPR technology by selectively reducing off-target editing. The laborious optimization of the engineered CRISPR inhibitors necessary by the time motivated us to more systematically investigate possibilities and constraints of protein engineering by domain insertion using an unbiased insertion approach. Previously, single protein domains were usually introduced only at a few rationally selected sites into target proteins. Here, we inserted up to five structurally and functionally unrelated domains into several different candidate effector proteins at all possible positions. The resulting libraries of protein hybrids were screened for activity by fluorescence-activated cell sorting (FACS) and subsequent next-generation sequencing (Flow-seq). Training machine learning models on the resulting, comprehensive datasets allowed us to dissect parameters that affect domain insertion tolerance and revealed that sequence conservation statistics are the most powerful predictors for domain insertion success. Finally, extending our experimental Flow-seq pipeline towards the screening of engineered, switchable effector variants yielded two potent optogenetic derivatives of the E. coli transcription factor AraC. These novel hybrids will enable the co-regulation of bacterial gene expression by light and chemicals. Taken together, our study showcases the design of functionally diverse protein switches for the control of gene editing and gene expression in mammalian cells and E. coli, respectively. In addition, the generation of a large domain insertion datasets enabled - for the first time - the unbiased investigation of domain insertion tolerance in several evolutionary unrelated proteins. Our study showcases the manifold opportunities and remaining challenges behind the engineering of proteins with new properties and functionalities by domain recombination

Maybe you can turn me on: CRISPRa-based strategies for therapeutic applications

Since the revolutionary discovery of the CRISPR-Cas technology for programmable genome editing, its range of applications has been extended by multiple biotechnological tools that go far beyond its original function as "genetic scissors". One of these further developments of the CRISPR-Cas system allows genes to be activated in a targeted and efficient manner. These gene-activating CRISPR-Cas modules (CRISPRa) are based on a programmable recruitment of transcription factors to specific loci and offer several key advantages that make them particularly attractive for therapeutic applications. These advantages include inter alia low off-target effects, independence of the target gene size as well as the potential to develop gene- and mutation-independent therapeutic strategies. Herein, I will give an overview on the currently available CRISPRa modules and discuss recent developments, future potentials and limitations of this approach with a focus on therapeutic applications and in vivo delivery

Synthetic biology to access and expand nature's chemical diversity

Bacterial genomes encode the biosynthetic potential to produce hundreds of thousands of complex molecules with diverse applications, from medicine to agriculture and materials. Accessing these natural products promises to reinvigorate drug discovery pipelines and provide novel routes to synthesize complex chemicals. The pathways leading to the production of these molecules often comprise dozens of genes spanning large areas of the genome and are controlled by complex regulatory networks with some of the most interesting molecules being produced by non-model organisms. In this Review, we discuss how advances in synthetic biology — including novel DNA construction technologies, the use of genetic parts for the precise control of expression and for synthetic regulatory circuits — and multiplexed genome engineering can be used to optimize the design and synthesis of pathways that produce natural products