Precise genome editing human stem cells for functional interrogation of genetic variation
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Date
2015-06-19
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Johns Hopkins University
Abstract
Human induced pluripotent stem cells (iPSCs) provide great potential as a tool for basic biological discovery, disease modeling, and ultimately for cell based regenerative medicine but the full realization of these stem cells requires an ability to precisely edit their genome in an efficient way. The relative efficiencies of CRISPR/Cas9 and TALENs were quantified in human iPSC lines for inducing both homologous donor-based precise genome editing and nonhomologous end joining (NHEJ)-mediated gene; genome wide off-target mutagenesis was also assessed by targeted deep sequencing. The specificity of Cas9 was further tested by targeting either the mutant or the wild-type allele of disease causing single nucleotide variants (SNVs) with gRNAs and testing their cleavage at the intended target or the other genotype differing by a single nucleotide; little disruption was observed at the other allele differing by a single nucleotide alone. Overall, these results demonstrate the advantages of the CRISPR/Cas9 system in allele-specific genome targeting and in NHEJ-mediated gene disruption while they were comparable in HDR efficiencies. To further investigate the specificity whole genome sequencing was performed on iPSCs successfully edited with either CRISPR/Cas9 or TALENs and found no evidence of mutations at sites similar to the nuclease binding sequence. To utilize these genome editing tools for a relevant biological phenotype genetic variation associated to platelet aggregation was chosen for further characterization. Platelet endothelial aggregation receptor 1 (PEAR1) was knocked out (KO) using CRISPR/Cas9 and compared to the otherwise isogenic iPSCs to determine its influence on hematopoiesis and megakaryopoiesis. PEAR1 KO iPSCs were found to increase cell proliferation as well as increase megakaryocyte lineage commitment. Additionally an intronic SNP, rs12041331, reported to reside within a putative enhancer was deleted along with the surrounding 251 bp which resulted in a significant reduction of PEAR1 mRNA levels. This reduction was primarily observed on the same allele as the deletion indicating a cis-acting mechanism of gene regulation. Overall this thesis demonstrates the efficiency and specificity of genome editing in human stem cells and their use towards precise genetic modification for functional interrogation of genetic variation.
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Keywords
iPSCs, CRISPR, Genome editing, disease modeling, TALEN, off-target, bioinformatics