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Principal Investigator: Le Cong
Organization: STANFORD UNIVERSITY
Fiscal Year: 2024
Award: $473,100
Funding agency: National Human Genome Research Institute
Project Abstract
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.
Terms: <Autoimmune Diseases><Bar Codes><Biologic Models><Biological Models><Black Box><CRISPR><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas system><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cancer Model><CancerModel><Cancers><Cas nuclease technology><Cell Body><Cells><Clustered Regularly Interspaced Short Palindromic Repeats><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Collection><Complex><Computational Technique><Computer Models><Computerized Models><DNA><DNA Recombination><Deoxyribonucleic Acid><Disease><Disorder><Drug resistance><Engineering><Epigenetic><Epigenetic Change><Epigenetic Mechanism><Epigenetic Process><Gene variant><Genetic><Genetic Recombination><Genome><Genome engineering><Goals><Human><Human Genome><In Vitro><Knowledge><Malignant Neoplasms><Malignant Tumor><Medicine><Methods><Modality><Model System><Modern Man><Modification><Nerve Degeneration><Neuron Degeneration><Non-Polyadenylated RNA><Proteins><RNA><RNA Gene Products><Recombination><Resolution><Ribonucleic Acid><Single Base Polymorphism><Single Nucleotide Polymorphism><System><Technology><Testing><Therapeutic><Toxic effect><Toxicities><Variant><Variation><Work><allele variant><allelic variant><autoimmune condition><autoimmune disorder><autoimmunity disease><barcode><cell type><computational modeling><computational models><computer based models><computerized modeling><design><designing><drug resistant><epigenetically><gene editing platform><gene editing system><gene editing technology><gene editing tools><gene locus><gene-editing toolkit><genetic locus><genetic variant><genome editing><genomic editing><genomic location><genomic locus><genomic variant><human disease><human whole genome><improved><in vivo><malignancy><mammalian genome><microbial><neoplasm/cancer><neural degeneration><neurodegeneration><neurodegenerative><neurological degeneration><neuronal degeneration><resistance to Drug><resistant to Drug><resolutions><single cell genomics><single nucleotide variant><success><tool><tumor>