Towards Robust Multiplex Genome Engineering Beyond CRISPR-Cas9 Exemplified by the CRISPR-Cas9 system, gene-editing technology is a powerful collection of tools for probing the hidden mechanisms of human diseases by understanding and controlling the functions of human genome variants. However, existing CRISPR genome technologies have three major limitations: (1) low efficiency and lack of accuracy when making large genome modifications such as structural variants in complex diseases; (2) uncontrollable off-target effects that lead to unwanted editing and cellular toxicity; (3) variable activity and precision when performing CRISPR editing in mammalian genome across different contexts, e.g. genomic loci, cell types, and model systems. To overcome these limitations, many groups including our own have sought to develop improved CRISPR tools using experimental methods and computational techniques. Building on my previous expertise, I will work towards multiplex, robust and error- free genome engineering. My group will seek to design new microbial proteins with sequence- independent recombination and RNA-to-DNA editing capabilities (Focus 1). Then, to provide robust gene-editing tools for studying single-cell genomics, I propose to leverage versatile CRISPR designs to enable high-capacity cell barcoding to define genome dynamics at single- cell resolution (Focus 2). To validate our new tools and as initial demonstration, we will use in human cancer models, with a focus on studying the cellular dynamics that lead to tumor drug resistance through genetic perturbation (Focus 3). The ultimate goal of my lab is to enable error- free engineering of genomic variants at any sizes, with robust activities across in vitro and in vivo applications. I will use this precise toolkit to uncover the functions of long genome alterations in human diseases, a major ?black box? in our genome. The success of the proposal has the promise to generate safe, reliable genome correction tools for therapeutics.

Public Health Relevance

Towards Multiplex Precision Genome Engineering Technology Beyond CRISPR As medicine advances to unravel mechanisms of complex human diseases such as cancer, auto-immune diseases, and neuro-degeneration, a remaining challenge is that our knowledge of the genetic and epigenetic basis of these disorders are often limited to small genome changes like single-nucleotide variants. The key bottleneck for uncover these genome black boxes is the lack the precision tools to study large genome changes. Thus, it demands new modalities of multiplexable and accurate genome engineering tools that we proposes to develop here, leveraging advances in computational modeling and genome editing, to push our ability to engineer, track, and control cellular genome, with cancer model as initial test ground.

National Institute of Health (NIH)
National Human Genome Research Institute (NHGRI)
Unknown (R35)
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Special Emphasis Panel (ZHG1)
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Fletcher, Colin F
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Stanford University
Schools of Medicine
United States
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