Juan Hurtado

Graduate Student

Co-Advised by John Dueber

Previous Education:

B.S. in Bioengineering at MIT

Project Title:

CRISPR-guided DNA Polymerases for Continuous Directed Evolution in Mammalian Cells


Natural selection demonstrates that genetically diverse populations evolve new traits that increase their fitness against selective pressure. Similarly, by introducing diverse genetic libraries into environments designed to favor the propagation of desired functionalities, we can emulate natural evolution to engineer useful new biotechnology. This technique is known as directed evolution and classically requires the iterative repetition of two separate steps: (i) constructing a diverse library of DNA coding sequences by random mutagenesis, and (ii) delivering those coding sequences into suitable living hosts for expression and screening for desired functionality.To minimize the frequency of false “winner” mutants whose success through screening does not ultimately translate to its intended use case, directed evolution screening pipelines must accurately recapitulate the environment in which variants are ultimately expected to function. Some therapeutic modalities, like immune cell therapies (e.g. CAR-T), may be improved by screening libraries of proteins directly within a human cell for their ability to initiate specific signalling cascades. Nevertheless, current directed evolution pipelines are unsuited for the repeated diversification and screening of genomically-integrated libraries in human cells. To address this need, I aim to adapt CRISPR-guided error-prone DNA polymerases (EvolvR) for the continuous, targeted diversification of transgenes in mammalian cells. By continuously generating diversity directly within the genome, EvolvR may eliminate screening bottlenecks related to inefficiencies in library delivery while enabling long-term diversification and interrogation of libraries for the discovery of rare and highly-optimized fitness peaks.