Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system.

Precise genetic modifications in model animals are essential for biomedical research. Here, we report a programmable "base editing" system to induce precise base conversion with high efficiency in zebrafish. Using cytidine deaminase fused to Cas9 nickase, up to 28% of site-specific single-base mutations are achieved in multiple gene loci. In addition, an engineered Cas9-VQR variant with 5'-NGA PAM specificities is used to induce base conversion in zebrafish. This shows that Cas9 variants can be used to expand the utility of this technology. Collectively, the targeted base editing system represents a strategy for precise and effective genome editing in zebrafish.The use of base editing enables precise genetic modifications in model animals. Here the authors show high efficient single-base editing in zebrafish using modified Cas9 and its VQR variant with an altered PAM specificity

CRISPR-Cas Changing Biology?

Eugene V. Koonin argues that fundamental research of CRISPR-Cas mechanisms has (among other things) illuminated “fundamental principles of genome manipulation.” Koonin's discussion provides important philosophical insights for how we should understand the significance of CRISPR-Cas systems. Yet the analysis he provides is only part of a larger story. There is also a human element to the CRISPR-Cas story that concerns its development as a technology. Accounting for this part of CRISPR's history reveals that the story Koonin provides requires greater nuance. I'll show how CRISPR-Cas technologies are not “natural” genome editing systems, but (in part) artifacts of human ingenuity. Furthermore, I'll argue that the story of CRISPR-Cas is not “primarily about research into fundamental biological mechanisms”, but is instead about the intertwining of applied and fundamental research programs

Connecting Sarcomere Protein Mutations to Pathogenesis in Cardiomyopathies: The Development of “Disease in a Dish” Models

Recent technological and protocol developments have greatly increased the ability to utilize stem cells transformed into cardiomyocytes as models to study human heart muscle development and how this is affected by disease associated mutations in a variety of sarcomere proteins. In this perspective we provide an overview of these emerging technologies and how they are being used to create better models of ‘disease in a dish’ for both research and screening assays. We also consider the value of these assays as models to explore the seminal processes in initiation of the disease development and the possibility of early interventions

Construction of non-canonical PAM-targeting adenosine base editors by restriction enzyme-free DNA cloning using CRISPR-Cas9

Molecular cloning is an essential technique in molecular biology and biochemistry, but it is frequently laborious when adequate restriction enzyme recognition sites are absent. Cas9 endonucleases can induce site-specific DNA double-strand breaks at sites homologous to their guide RNAs, rendering an alternative to restriction enzymes. Here, by combining DNA cleavage via a Cas9 endonuclease and DNA ligation via Gibson assembly, we demonstrate a precise and practical DNA cloning method for replacing part of a backbone plasmid. We first replaced a resistance marker gene as a proof of concept and next generated DNA plasmids that encode engineered Cas9 variants (VQR, VRER and SpCas9-NG), which target non-canonical NGA, NGCG and NG protospacer-adjacent motif (PAM) sequences, fused with adenosine deaminases for adenine base editing (named VQR-ABE, VRER-ABE and NG-ABE, respectively). Ultimately, we confirmed that the re-constructed plasmids can successfully convert adenosine to guanine at endogenous target sites containing the non-canonical NGA, NGCG and NG PAMs, expanding the targetable range of the adenine base editing.This work was supported by National Research Foundation of Korea (NRF) Grants (no. 2018M3A9H3022412), Next Generation BioGreen 21 Program grant no. PJ01319301, Technology Innovation Program funded by the Ministry of Trade, Industry and Energy (no. 20000158), and Korea Healthcare technology R&D Project grant no. HI16C1012 to S.B

Master of Science

thesisHIV-1 latently infected cells are the major hurdle impeding viral eradication despite the development of ART (Anti-retroviral therapy), which works by inhibiting various viral proteins necessary for HIV-1 replication. Even after years of daily regimens of ART therapy, HIV-1 reemerges once the ART is discontinued. This is because HIV-1 can go latent or quiescent in resting CD4+ cells. These resting CD4+ cells contain integrated HIV DNA within the genetic material in the host cell, but no viral proteins are produced, and they are thus immune to circulating antiretroviral drugs. For that purpose, it is essential to understand the mechanisms and genes involved in the development, maintenance, and activation of latency. To investigate functions of transcripts and pathways critical for biological processes and disease mechanisms, gene knockout is a very useful technique. We propose to use the CRISPR/ cas9 system to knockout target genes and test if these genes are involved in the development of latency or are involved in the reactivation of latently infected cell

Induced Pluripotent Stem Cells Meet Genome Editing

It is extremely rare for a single experiment to be so impactful and timely that it shapes and forecasts the experiments of the next decade. Here, we review how two such experiments - the generation of human induced pluripotent stem cells (iPSCs) and the development of CRISPR/Cas9 technology - have fundamentally reshaped our approach to biomedical research, stem cell biology, and human genetics. We will also highlight the previous knowledge that iPSC and CRISPR/Cas9 technologies were built on as this groundwork demonstrated the need for solutions and the benefits that these technologies provided and set the stage for their success.National Institutes of Health (U.S.) (Grant 1R01NS088538-01)National Institutes of Health (U.S.) (Grant 2R01MH104610-15

Genomic and proteomic biases inform metabolic engineering strategies for anaerobic fungi.

Anaerobic fungi (Neocallimastigomycota) are emerging non-model hosts for biotechnology due to their wealth of biomass-degrading enzymes, yet tools to engineer these fungi have not yet been established. Here, we show that the anaerobic gut fungi have the most GC depleted genomes among 443 sequenced organisms in the fungal kingdom, which has ramifications for heterologous expression of genes as well as for emerging CRISPR-based genome engineering approaches. Comparative genomic analyses suggest that anaerobic fungi may contain cellular machinery to aid in sexual reproduction, yet a complete mating pathway was not identified. Predicted proteomes of the anaerobic fungi also contain an unusually large fraction of proteins with homopolymeric amino acid runs consisting of five or more identical consecutive amino acids. In particular, threonine runs are especially enriched in anaerobic fungal carbohydrate active enzymes (CAZymes) and this, together with a high abundance of predicted N-glycosylation motifs, suggests that gut fungal CAZymes are heavily glycosylated, which may impact heterologous production of these biotechnologically useful enzymes. Finally, we present a codon optimization strategy to aid in the development of genetic engineering tools tailored to these early-branching anaerobic fungi