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Principal Investigator: Yi Yin
Organization: UNIVERSITY OF CALIFORNIA LOS ANGELES
Fiscal Year: 2024
Award: $390,000
Funding agency: National Institute of General Medical Sciences
PROJECT SUMMARY/ABSTRACT
Homologous recombination (HR) is important for repairing DNA double strand breaks (DSB), and thus is an
essential process in embryonic development, meiosis and suppressing tumorigenesis. HR can also be a double-
edged sword: unrepaired breaks lead to cell death; errors by HR, particularly the crossover type, can cause
extensive genome rearrangements. While the biochemical process of HR in the repair of an induced DSB has
been elucidated by various methods, two critical gaps remain regarding spontaneous HR: 1) the lesions driving
spontaneous mitotic HR (e.g. in addition to DSBs, template switching in replication initiated by single-strand nicks
can also promote HR); and 2) HR partner choice that determines whether HR is error-free or not. The two gaps
are inter-related as HR partner choice could depend on the types of initiating lesions. What determines whether
HR is error-free or not is a fundamental question of what governs genome integrity. A major technical roadblock
is the lack of scalable, genome-wide tools for studying rare spontaneous HR events in many mutants. To scale
up genome-wide HR mapping efforts, I developed sci-L3, which enables linear amplification of single-cell
genomes that scales to 1M cells, enabling generating hundreds of single-cell global HR maps per mutant for
thousands of mutants. Moreover, genetic assays require detecting “scars” in the genome as traces of repair (e.g.
in cancer mutational signature studies), which miss 99% of error-free HR between identical sister chromatids.
There is thus a critical need for unbiased global assays that detect both mutational and error-free HR. I recently
addressed this gap by developing sci-L3-Strand-seq as the first scalable mapping tool for HR between identical
sister chromatids. Our central vision is to determine how spontaneous mitotic crossovers cause genome
rearrangements by scalable single-cell assays. In Area1, we will use sci-L3 to explore the full mutant space
in hybrid yeast diploids. By generating HR maps in all the single mutants in a pooled manner (160 single-cell HR
maps/mutant for 6,000 mutants), we can simultaneously test and generate thousands of hypotheses regarding
different lesions and pathways that drive different types of genome instability events genome-wide. In Area2, we
focus on a deciding factor for whether HR is error-free or not: HR partner choice of allelic sister chromatid, allelic
homolog and non-allelic repeats. With sci-L3-Strand-seq, we propose to map all the seven classes of crossover
outcomes in two systems: mammalian cell lines and mouse embryos. In cell lines, we will investigate genome-
wide distributions of both error-free and mutational HR outcomes in the wild-type as well as hundreds of
perturbations of HR-related genes to determine factors affecting HR partner choice including (epi)genomic
contexts, 3D genome organization and HR gene knockdown. We will also develop in vivo sci-L3-Strand-seq/RNA
co-assay to dissect cell-type variation in HR partner choice in mouse embryos. In sum, this proposal pursues
cost-effective, massively parallel and genome-wide mapping of mitotic crossover outcomes as functions of
genetic perturbations and cell types by developing and applying advanced single-cell multi-omics tools.
Terms: <3-D><3-Dimensional><3D><Address><Affect><Alleles><Allelomorphs><Area><Assay><Automobile Driving><Bioassay><Biochemical Process><Biological Assay><Body Tissues><Cancers><Cell Body><Cell Death><Cell Line><CellLine><Cells><Cellular Assay><Cicatrix><DNA><DNA Alteration><DNA Damage Repair><DNA Double Strand Break><DNA Repair><DNA Replication Initiation><DNA Sequence Alteration><DNA mutation><Deoxyribonucleic Acid><Diploid><Diploidy><Embryo><Embryo Development><Embryogenesis><Embryonic><Embryonic Development><Event><GeneHomolog><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic defect><Genetic mutation><Genome><Genome Instability><Genomic Instability><Genomics><Homolog><Homologous Gene><Homologue><Human><Hybrids><Lead><Lesion><Malignant Neoplasms><Malignant Tumor><Mammalian Cell><Maps><Meiosis><Methods><Mice><Mice Mammals><Mitotic><Modern Man><Murine><Mus><Mutation><Non-Polyadenylated RNA><Oncogenesis><Outcome><Pathway interactions><Pb element><Process><RNA><RNA Gene Products><Replication Initiation><Ribonucleic Acid><Scars><Sequence Alteration><Sight><Single cell seq><Sister Chromatid><Strains Cell Lines><System><Technology><Testing><Tissues><Unscheduled DNA Synthesis><Variant><Variation><Vision><Yeasts><cell assay><cell type><cost effective><cultured cell line><detection assay><driving><experience><genome integrity><genome mutation><genome scale><genome-wide><genomewide><genomic alteration><genomic integrity><heavy metal Pb><heavy metal lead><homologous recombination><in vivo><knock-down><knockdown><malignancy><meiotic><multiomics><multiple omics><mutant><necrocytosis><neoplasm/cancer><panomics><pathway><repair><repaired><scale up><single cell next generation sequencing><single cell sequencing><three dimensional><tool><tumorigenesis><visual function>