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Project 1. Genome engineering by designed structural folding

To generate Cas9 systems with new or enhanced functionality, numerous protein engineering strategies have been employed by other research teams. Methods like in vitro evolution or structure-based predictions are known to increase precision and robustness of gene editing by introducing mutations into proteins. Nevertheless, Cas9 modification via mutagenesis has yet not reached its full potential, since most of the proposals have been limited to amino acid substitutions.

In nature, the Cas9 protein exists in various molecular sizes, while minimally affecting its intended activity level. Nature has profusely inserted or deleted amino acids during the Cas9 protein evolution, rather resulting in a plethora of orthologues. Therefore, we need an integrated approach of molecular modeling, functional verification, and structure validation to create desirable versions of Cas9.

We have assembled a team of relevant experts to address these limitations. Dr. Minkyung Baek (Seoul National University), the lead developer of the innovative protein structure prediction program RoseTTAFold, is our main collaborator on the project. Dr. Ji-Hye Yun (CGE) has been recruited for her expertise in protein structural biology, supported by numerous publications using NMR, crystallography, X-FEL, and Cryo-EM. Furthermore, Dr. Annie Kim’s team (CGE) and Dr. Kayeong Lim (KIST) will confirm the acceptability of newly designed Cas9 candidates.

Project 2. Borderless gene editing

CRISPR/Cas9 is becoming the general way to model mutant animals and plants, especially for experimental purposes. The technology has deeply advanced its use in mice models as it is able to generate targeted gene knockout, knockin, and even conditional gene knockout with some levels of complexity. Expanding this technological advancement to a wider spectrum of species will innovate agricultural, livestock, and marine strains developments and will also present animal models that better represent human diseases.

The application of CRISPR/Cas9 advancements to new model species remains challenging at this point and researchers are limited to a handful of conventional organismal models. Our team has launched an ambitious project to establish DNA transfer to organisms in a borderless manner and expand gene editing methods into as yet unexplored species.

The unique agricultural and marine environment of South Korea is highly suited to this research, making the center a highly suitable place for a leading research hub in the field. The country’s historic aquaculture communities has collected valuable information on the life cycles, breeding, and development in various marine species, which can be transferred from bench to bedside.

Furthermore, South Korea also has a long-standing tradition of medicinal herbs, many of which are commercially local. For this project, we are bringing together a team of scientists to develop specific genome engineering methods, with a primary focus on gene transfer for fish and herb species grown in South Korea.

Project 3. Short conditional intrON (SCON) & inverse short conditional intrON (ICON)

SCON (short conditional intron) & ICON (inverse short conditional intron)

Reliable animal models are critical for understanding disease and developing therapeutics. CRISPR/Cas9-based gene editing has opened new opportunities for disease modeling, such as hereditary diseases with late-onset symptoms. Gene knockouts and knock-ins into mice models have become possible with ease. However, even with the introduction of CRISPR/Cas9, conditional gene modification still remains challenging

To address this problem, we are working with two novel strategies that each utilize a unique design of short conditional introns: SCON and ICON. The former is designed to generate conditional knockout alleles (WT to KO), whereas the latter is designed to revert knockout alleles to normal functional alleles (KO to WT). The methods require a simple zygotic injection at the one- or two-cell stage, so any vertebrate that has a feasible approach to the fertilized egg can become a subject model (Wu et al., EMM, 2022).

Project 4. Mosaic genetics

This project contributes to a new field of mosaic genetics by tailoring the Rosa-Confetti multicolor reporter cassette so that any cDNAs of interest are co-expressed with red fluorescence. This method allows functional genetics to be performed at a clonal level with the outcomes being accessible to quantitative clonal analysis, thick tissue imaging, fluorescence-activated cell sorting, and single-cell RNA-sequencing.

Our primary goal with this project is to reveal the characteristics of mutant clones and their microenvironment in early oncogenesis. We are looking into the prime gene regulations and cellular fundamentals during the earliest stage of neoplastic transformation. To date, this pathologic period has been difficult to follow and investigate, due to the lack of appropriate genetic tools. Utilizing this method, we have been able to determine that oncogenic signals assign scattered, pre-malignant clone dominance to their wild-type neighbor clones within the intestinal epithelium [Red2Onco system; Yum et al., Nature, 2021 (12)]. Furthermore, we are expanding our research to tumor suppressors and by modifying this model to obtain clonal knockout capabilities. Potential therapeutic intervention strategies are being investigated in depth by this model as well.