Jae Keith Joung
The Joung laboratory is developing strategies to reprogram the genome and epigenome of living cells to better understand biology and treat disease. We have developed and optimized molecular tools for customized genome editing that enable scientists to alter the DNA sequence of a living cell—from fruit flies to humans—with great precision. These technologies are based on proteins engineered to recognize and cleave specific genomic sequences. We also use these targeting methodologies to enable activation, repression, or alteration of histone modifications of specific genes. These tools have many potential research uses and may one day lead to more efficient gene therapy capable of correcting disease-related mutations in human cells.
Genome Editing Using Targeted Nucleases
Genome editing technology using CRISPR-Cas nucleases was recently named “Breakthrough of the Year” for 2015 by Science magazine. Much of our recent work with genome-editing nucleases has focused on CRISPR-Cas9 and CRISPR-Cpf1(Cas12a). We and our collaborators were the first to demonstrate that these nucleases can function to modify single-cell embryos in vivo (Hwang & Fu et al., Nat Biotechnol. 2013), modifying endogenous genes in zebrafish embryos. We also were the first to show that Cas9 nucleases can induce significant off-target mutations in human cells (Fu et al., Nat Biotechnol. 2013). We developed GUIDE-seq, an unbiased, genome-wide method for sensitive detection of CRISPR-Cas9-induced off-target mutations in human cells (Tsai et al., Nat Biotechnol. 2015) that is now widely used in the field, and CIRCLE-seq, a highly sensitive in vitro method for defining genome-wide off-target cleavage sites of CRISPR-Cas9 nucleases (Tsai et al., Nat Methods 2017). Using GUIDE-seq, we have defined the genome-wide specificities of CRISPR-Cpf1(Cas12a) nucleases (Kleinstiver et al., Nat Biotechnol. 2016). Using structure-guided design, we have engineered “high-fidelity” Cas9 variants that robustly fail to show detectable genome-wide off-targets as judged by GUIDE-seq (Kleinstiver & Pattanayak et al., Nature 2016). Finally, we used a combination of structure-guided design and molecular evolution to engineer Cas9 variants with novel DNA binding specificities, thereby broadening the targeting range and applications of this platform (Kleinstiver et al., Nature 2015; Kleinstiver et al., Nat Biotechnol. 2015).
Epigenome Editing Using Targeted Transcription Factors
We have also demonstrated that the TALE and CRISPR platforms can also be utilized to create artificial transcription factors that can robustly alter expression of endogenous human genes (Maeder et al., Nat Methods 2013a; Maeder et al., Nat Methods 2013b; Tak et al., Nat Methods 2017). In addition, we have collaborated with Brad Bernstein's group to develop fusions of the histone demethylase LSD1 with TALE domains that can induce targeted histone alterations at endogenous human enhancers (Mendenhall et al., Nat Biotechnol. 2013). Finally, we have also developed fusions of engineered TALE domains with the catalytic domain of the TET1 enzyme, enabling the targeted demethylation of CpGs in human cells (Maeder et al., Nat Biotechnol. 2013).
Molecular Pathology Unit
149 13th St., 6th Floor
Charlestown, MA 02129