Abstract
Recent advances allow multiplexed genome engineering in E. coli, employing easily designed oligonucleotides to edit multiple loci simultaneously. A similar technology in human cells would greatly expedite functional genomics, both by enhancing our ability to test how individual variants such as single nucleotide polymorphisms (SNPs) are related to specific phenotypes, and potentially allowing simultaneous mutation of multiple loci. However, oligo-mediated targeting of human cells is currently limited by low targeting efficiencies and low survival of modified cells. Using a HeLa-based EGFP-rescue reporter system we show that use of modified base analogs can increase targeting efficiency, in part by avoiding the mismatch repair machinery. We investigate the effects of oligonucleotide toxicity and find a strong correlation between the number of phosphorothioate bonds and toxicity. Stably EGFP-corrected cells were generated at a frequency of ~0.05% with an optimized oligonucleotide design combining modified bases and reduced number of phosphorothioate bonds. We provide evidence from comparative RNA-seq analysis suggesting cellular immunity induced by the oligonucleotides might contribute to the low viability of oligo-corrected cells. Further optimization of this method should allow rapid and scalable genome engineering in human cells.
Highlights
New sequencing technologies are producing a wealth of data, and soon it will be possible to sequence full human genomes for about $1000 [1,2]
Selection markers or residual recombinase sequences could confound subtle phenotypes such as those caused by variants in regulatory regions, which are of particular interest since expression variability of,5% of all human genes is linked to single-nucleotide polymorphisms (SNPs) located within 200 kb of the gene [5]
The EGFP+ population was estimated by allele-specific qPCR (AS-qPCR) to carry 11–13% converted DNA, which matches the 12.5% expected if these cells have undergone a single oligo incorporation at one of the two genomic modified EGFP gene (mEGFP) during DNA replication (1/8 strands at the end of S phase) but not yet proceeded through cell division
Summary
New sequencing technologies are producing a wealth of data, and soon it will be possible to sequence full human genomes for about $1000 [1,2]. Genome-wide association studies (GWAS) have revealed many single-nucleotide polymorphisms (SNPs) linked to disease phenotypes, most of which are ‘tag SNPs’ in the context of large genomic regions where the actual causal variants remain unknown [3]. As of this writing, NHGRI’s GWAS Catalog contains 6,030 SNPs with a p-value ,1.061025. Selection markers or residual recombinase sequences could confound subtle phenotypes such as those caused by variants in regulatory regions, which are of particular interest since expression variability of ,5% of all human genes is linked to SNPs located within 200 kb of the gene [5]
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