Human health is intrinsically linked to the health of the indigenous microbial ecosystems living in the skin, the mouth, and especially the gastrointestinal tract. While deep sequencing has provided a window into the composition of these communities, we currently lack robust and targeted methods of intervention. With few exceptions, this dearth of techniques prevents us from restoring unbalanced ecosystems to a healthy state, immunizing the microbiota against further disruption, or even investigating the roles played by particular species or genes by perturbing them and observing the effects on the wider community. This proposal seeks to harness the CRISPR-Cas acquired immune system to control the abundance of specific genes and bacteria within the microbiota for the study and in situ treatment of enteric disease. I have shown that cells engineered to degrade bacteriophage genes are able to outcompete susceptible bacteria in the presence of phages, and also that mobile genetic elements engineered to degrade pathogenic genes can immunize bacterial populations as they spread. Because phages and mobile elements can be highly specific, these innovations can control the abundance of targeted genes and species without affecting unrelated microbes in mixed cultures. During the K99 phase, I will learn to work with animal models and pathogenic bacteria in order to test the efficacy of these methods in vivo. Specifically, I will seek to 1) stably replace potentially harmful microbes with protective strainsin the mouse gut, and 2) spread Cas9-mediated immunity through the native microbiota using mobile genetic elements that copy their payload into the host genome. Having acquired new skills and explored the capabilities of these novel technologies, I will apply them during the R00 phase to 3) treat and prevent Shiga toxin-mediated enteric disease by eliminating the toxin-encoding genes with mobile Cas9 elements and by colonizing the gut with protective strains, and 4) reduce inflammation in mice using stable populations of microbes engineered to secrete consistent levels of anti-inflammatory molecules in close proximity to the inflamed epithelium. I expect these innovative approaches to illuminate the role of the microbiota in human health and establish a new basis for the in situ treatment and prevention of enteric disease.
Human health is the result of cooperation between our own cells and the complex microbial communities living within us. Disrupted and unbalanced microbial ecosystems are more vulnerable to infection and have also been linked to diabetes, obesity, immune disorders, and cancer. We propose to harness the newly discovered bacterial immune system to restore community balance, immunize the ecosystem against future disruptions, and strengthen the regulatory signals normally employed by the body and its microbial inhabitants to address digestive and nutritional disorders from within.
|DiCarlo, James E; Chavez, Alejandro; Dietz, Sven L et al. (2015) Safeguarding CRISPR-Cas9 gene drives in yeast. Nat Biotechnol 33:1250-1255|
|Yaung, Stephanie J; Esvelt, Kevin M; Church, George M (2015) Complete Genome Sequences of T4-Like Bacteriophages RB3, RB5, RB6, RB7, RB9, RB10, RB27, RB33, RB55, RB59, and RB68. Genome Announc 3:|
|Yaung, Stephanie J; Esvelt, Kevin M; Church, George M (2014) CRISPR/Cas9-mediated phage resistance is not impeded by the DNA modifications of phage T4. PLoS One 9:e98811|