Person: Lin, Chieyu
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Chieyu
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Lin, Chieyu
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Publication Characterization and Optimization of the CRISPR/Cas System for Applications in Genome Engineering(2014-07-07) Lin, ChieyuThe ability to precisely manipulate the genome in a targeted manner is fundamental to driving both basic science research and development of medical therapeutics. Until recently, this has been primarily achieved through coupling of a nuclease domain with customizable protein modules that recognize DNA in a sequence-specific manner such as zinc finger or transcription activator-like effector domains. Though these approaches have allowed unprecedented precision in manipulating the genome, in practice they have been limited by the reproducibility, predictability, and specificity of targeted cleavage, all of which are partially attributable to the nature of protein-mediated DNA sequence recognition. It has been recently shown that the microbial CRISPR-Cas system can be adapted for eukaryotic genome editing. Cas9, an RNA-guided DNA endonuclease, is directed by a 20-nt guide sequence via Watson-Crick base-pairing to its genomic target. Cas9 subsequently induces a double-stranded DNA break that results in targeted gene disruption through non-homologous end-joining repair or gene replacement via homologous recombination. Finally, the RNA guide and protein nuclease dual component system allows simultaneous delivery of multiple guide RNAs (sgRNA) to achieve multiplex genome editing with ease and efficiency. The potential effects of off-target genomic modification represent a significant caveat to genome editing approaches in both research and therapeutic applications. Prior work from our lab and others has shown that Cas9 can tolerate some degree of mismatch with the guide RNA to target DNA base pairing. To increase substrate specificity, we devised a technique that uses a Cas9 nickase mutant with appropriately paired guide RNAs to efficiently inducing double-stranded breaks via simultaneous nicks on both strands of target DNA. As single-stranded nicks are repaired with high fidelity, targeted genome modification only occurs when the two opposite-strand nicks are closely spaced. This double nickase approach allows for marked reduction of off-target genome modification while maintaining robust on-target cleavage efficiency, making a significant step towards addressing one of the primary concerns regarding the use of genome editing technologies. The ability to multiplex genome engineering by simply co-delivering multiple sgRNAs is a versatile property unique to the CRISPR-Cas system. While co-transfection of multiple guides is readily feasible in tissue culture, many in vivo and therapeutic applications would benefit from a compact, single vector system that would allow robust and reproducible multiplex editing. To achieve this, we first generated and functionally validated alternate sgRNA architectures to characterize the structure-function relationship of the Cas9 protein with the sgRNA in DNA recognition and cleavage. We then applied this knowledge towards the development and optimization of a tandem synthetic guide RNA (tsgRNA) scaffold that allows for a single promoter to drive expression of a single RNA transcript encoding two sgRNAs, which are subsequently processed into individual active sgRNAs.Publication Developmental Origins of Aggressive Medulloblastoma(2013-03-05) Lin, Chieyu; Jackson-Grusby, Laurie Lynne; Daley, George; He, Xi; Elledge, StephenMedulloblastomas represent a heterogeneous group of cerebellar tumors that constitute the most frequent primary pediatric solid malignancy. Molecular characterization of these tumors have led to the understanding that distinctsubtypes possess characteristic properties such as gene expression profile, histological classification, and degree of dissemination that are predictive of disease progression and prognosis. Fractionation of primary medulloblastomas has led to the appreciation of brain tumor stem cells (BTSC) that may be driving the more aggressive and malignant disease. However, the developmental origins of these cells as well as the influences of early mutations in tumor suppressors on development and tumorigenesisremain unclear. My work is geared towards understanding the impact of mutations in the key tumor suppressor genes Ptc1 and p53 on medulloblastoma formation. I first identified key differences in neural stem cell marker expression that distinguish between Ptc1 and Ptc1;p53 medulloblastomas, demonstrating that the Ptc1;p53 genotype may pre-dispose to a more malignant, stem-like tumor. Through the use of a somatic mosaic model, we describe a synergistic interaction between Ptc1 haploinsufficiency and p53 deficiency leading to developmental seeding of the cerebellar field by pre-malignant cells and term this phenomenon “developmental field cancerization.” Interestingly, we observed this premalignant colonization in the cerebellarstem cell compartment as well, resulting in an aberrant population of self-renewing cells. Upon loss-of-heterozygosity at the Ptc1 locus, the Ptc;p53 animals alone develop robust cerebellar tumorsthat possess a definable stem-like population of cells that can re-initiate metastatic secondary tumors. These findings demonstrate how early mutationsin the tumor suppressor genes, such as Ptc1 and p53, may lead to stem cell field cancerization and play an important role in determining future tumor character and prognosis.Publication Shifts in receptors during submergence of an encephalitic arbovirus(Springer Science and Business Media LLC, 2024-07-24) Li, Wanyu; Plante, Jessica A.; Lin, Chieyu; Basu, Himanish; Plung, Jesse S.; Fan, Xiaoyi; Boeckers, Joshua M.; Oros, Jessica; Buck, Tierra K.; Anekal, Praju V.; Hanson, Wesley A.; Varnum, Haley; Wells, Adrienne; Mann, Colin J.; Tjang, Laurentia V.; Yang, Pan; Reyna, Rachel A.; Mitchell, Brooke M.; Shinde, Divya P.; Walker, Jordyn L.; Choi, So Yoen; Brusic, Vesna; Montero Llopis, Paula; Weaver, Scott C.; Umemori, Hisashi; Chiu, Isaac M.; Plante, Kenneth S.; Abraham, JonathanWestern equine encephalitis virus (WEEV) is an arthropod-borne virus (arbovirus) that frequently caused major outbreaks of encephalitis in humans and horses in the early 20th century, but outbreak frequency has since decreased drastically, and strains of this alphavirus isolated in the last two decades are less virulent in mammals than strains isolated in the 1930s–40s. The basis for this phenotypic change in WEEV strains and coincident decrease in epizootic activity (“viral submergence") is unclear, as is the possibility of re-emergence of highly virulent strains. Here, we identified protocadherin 10 (PCDH10), as a cellular receptor for WEEV. We show that multiple highly virulent ancestral WEEV strains isolated in the 1930s–1940s, in addition to binding human PCDH10, could also bind very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), which are recognized by another encephalitic alphavirus as receptors. However, while most of the WEEV strains we examined bind to PCDH10, a contemporary strain lost the ability to recognize mammalian PCDH10 while retaining the ability to bind avian receptors, suggesting WEEV adaptation to a main reservoir host during enzootic circulation. PCDH10 supports WEEV E2–E1-mediated infection of primary murine cortical neurons and administration of a soluble form of PCDH10 protects mice from lethal WEEV challenge. Our results have implications for the development of medical countermeasures and for risk assessment for re-emerging WEEV strains.