Rapid Prototyping and Analysis of Targeted Nuclease TechnologiesView all posters
MIT, United States
Genome engineering technologies such as ZFNs, TALENs, and CRISPR/Cas systems have the potential to revolutionize therapeutic strategies for disease intervention and accelerate the creation of designer genetic systems, with applications ranging from agriculture to neuroscience.
We recently created prototyping pipeline for the rapid design of single and multiplexed CRISPR/Cas systems with target specificities optimized over the entire genome. Results from our lab and others have shown that the specificity of the Streptococcus pyogenes CRISPR/Cas Type II system in mammalian cells varies as a function of the guide RNA sequence used to target the Cas9 nuclease to a specific gene. We constructed large oligo libraries of guide RNAs carrying combinations of mutations to study the sequence dependence of Cas9 programming. Data from these studies were used to develop algorithms for the prediction of CRISPR/Cas off-target activity across the human genome. Our resulting computational platform supports the prediction of specificity for CRISPR/Cas systems from any species.
Additionally, we developed a next-generation deep sequencing (NGS) and data processing pipeline for rapidly assessing the on-target gene editing performance and genome-wide off-target activity of any targeted nuclease technology. We used the Ion Torrent platform to sequence genomic regions flanking target loci and predicted off-target sites across the human genome. We present a library of computational tools for processing NGS sequencing data and detecting indels. The combination of our CRISPR prototyping and NGS sequencing pipeline resources will greatly advance the rational design and precise application of genome engineering technologies.