The Center for Transcriptional Consequences of Human Genetic Variation (CTCHGV) will develop innovative and powerful genetic engineering methods and use them to identify genetic variations that causally control gene transcription levels. Genome Wide Association Studies (GWAS) find many variations associated with disease and other phenotypes, but the variations that may actually cause these conditions are hard to identify because nearby variations in the same haplotype blocks consistently co-occur with them in human populations, so that specifically causative ones cannot be distinguished. About 95% of GWAS variations are not in gene coding regions, and many of these presumably associate with altered gene expression levels. CTCHGV will identify the variations that directly control gene expression by engineering precise combinations of changes to gene regulatory regions that break down the haplotype blocks, allowing each variations'effect on gene expression to be discerned independently of the others. To perform this analysis, CTCHGV will extract ~100kbps gene regulatory regions from human cell samples, create precise variations in them in E. coli, and re-introduce the altered regions back into human cells, using zinc finger nucleases (ZFNs) to efficiently induce recombination. CTCHGV will target 1000 genes for this analysis (Aim 1), and will use human induced Pluripotent Stem cells (iPS) to study the effects of variations in diverse human cell types (Aim 2). To explore the effects of variations in complex human tissues, CTCHGV will develop methods of measuring gene expression at transcriptome-wide levels in many single cells, including in situ in structured tissues (Aim 3). Finally, CTCHGV will develop novel advanced technologies that integrate DNA sequencing and synthesis to construct thousands of large DNA constructs from oligonucleotides, that enable very precise targeting and highly efficient performance of ZFNs, and that enable cells to be sorted on the basis of morphology as well as fluorescence and labeling (Aim 4). CTCHGV will also develop direct oligo-mediated engineering of human cells, and create """"""""marked allele"""""""" iPS that will enable easy ascertainment of complete exon distributions for many pairs of gene alleles in many cell types.

Public Health Relevance

CTCHGV methods will yield precise knowledge of effects of human genetic variations on gene expression that will both refine and go beyond GWAS-derived associations between non-coding variations and disease. Powerful new CTCHGV genetic engineering methods will directly enable gene therapy. CTCHGV iPS and single-cell transcriptome technologies will increase understanding of diverse and complex human tissues.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Specialized Center (P50)
Project #
3P50HG005550-04S1
Application #
8792903
Study Section
Ethical, Legal, Social Implications Review Committee (GNOM)
Program Officer
Schloss, Jeffery
Project Start
2010-09-13
Project End
2015-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
4
Fiscal Year
2014
Total Cost
$35,966
Indirect Cost
$14,727
Name
Harvard University
Department
Genetics
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Bester, Assaf C; Lee, Jonathan D; Chavez, Alejandro et al. (2018) An Integrated Genome-wide CRISPRa Approach to Functionalize lncRNAs in Drug Resistance. Cell 173:649-664.e20
Guo, Xiaoge; Chavez, Alejandro; Tung, Angela et al. (2018) High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR-Cas9 in yeast. Nat Biotechnol 36:540-546
Chavez, Alejandro; Pruitt, Benjamin W; Tuttle, Marcelle et al. (2018) Precise Cas9 targeting enables genomic mutation prevention. Proc Natl Acad Sci U S A 115:3669-3673
Yeo, Nan Cher; Chavez, Alejandro; Lance-Byrne, Alissa et al. (2018) An enhanced CRISPR repressor for targeted mammalian gene regulation. Nat Methods 15:611-616
Chan, Yingleong; Chan, Ying Kai; Goodman, Daniel B et al. (2018) Enabling multiplexed testing of pooled donor cells through whole-genome sequencing. Genome Med 10:31
Kalhor, Reza; Kalhor, Kian; Mejia, Leo et al. (2018) Developmental barcoding of whole mouse via homing CRISPR. Science 361:
Chari, Raj; Yeo, Nan Cher; Chavez, Alejandro et al. (2017) sgRNA Scorer 2.0: A Species-Independent Model To Predict CRISPR/Cas9 Activity. ACS Synth Biol 6:902-904
Niu, Dong; Wei, Hong-Jiang; Lin, Lin et al. (2017) Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science 357:1303-1307
Aach, John; Lunshof, Jeantine; Iyer, Eswar et al. (2017) Correction: Addressing the ethical issues raised by synthetic human entities with embryo-like features. Elife 6:
Shipman, Seth L; Nivala, Jeff; Macklis, Jeffrey D et al. (2017) CRISPR-Cas encoding of a digital movie into the genomes of a population of living bacteria. Nature 547:345-349

Showing the most recent 10 out of 91 publications