Research Highlights

I earned my Ph.D. in chemistry and chemical biology from Harvard University, in the lab of David R Liu. The lab specializes in the evolution of proteins and small molecules for therapeutic and genome-editing applications. Below are highlights of my research.

 
 
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High-resolution specificity profiling and off-target prediction for site-specific DNA recombinases

Jeffrey L Bessen, Lena K Afeyan, Vlado Dančík, Luke W Koblan, David B Thompson, Chas Leichner, Paul A Clemons, and David R Liu. Nature Communications (2019), DOI: 10.1038/s41467-019-09987-0

ABSTRACT: The development of site-specific recombinases (SSRs) as genome editing agents is limited by the difficulty of altering their native DNA specificities. Here we describe Rec-seq, a method for revealing the DNA specificity determinants and potential off-target substrates of SSRs in a comprehensive and unbiased manner. We applied Rec-seq to characterize the DNA specificity determinants of several natural and evolved SSRs including Cre, evolved variants of Cre, and other SSR family members.

 
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Rec-seq profiling of these enzymes and mutants thereof revealed previously uncharacterized SSR interactions, including specificity determinants not evident from SSR:DNA structures. Finally, we used Rec-seq specificity profiles to predict off-target substrates of Tre and Brec1 recombinases, including endogenous human genomic sequences, and confirmed their ability to recombine these off-target sequences in human cells. These findings establish Rec-seq as a high-resolution method for rapidly characterizing the DNA specificity of recombinases with single-nucleotide resolution, and for informing their further development.

 
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A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells 

Brian Chaikind, Jeffrey L Bessen, David B Thompson, Johnny H Hu, and David R Liu. Nucleic Acids Research (2016), 44, 9758-70.

ABSTRACT: We describe the development of 'recCas9', an RNA-programmed small serine recombinase that functions in mammalian cells. We fused a catalytically inactive dCas9 to the catalytic domain of Gin recombinase using an optimized fusion architecture. The resulting recCas9 system recombines DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences. We show that these recombinases can operate on DNA sites in mammalian cells identical to genomic loci naturally found in the human genome in a manner that is dependent on the guide RNA sequences.

 
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DNA sequencing reveals that recCas9 catalyzes guide RNA-depended recombination in human cels with an efficiency as high as 32% on plasmid substrates. Finally, we demonstrate that recCas9 expressed in human cells can catalyze in situ deletion between two genomic sites. Because recCas9 directly catalyzes recombination, it generates virtually no detectable indels or other stochastic DNA modification products. This work represents a step toward programmable, scarless genome editing in unmodified cells that is independent of endogenous machinery or cell state. Current and future generations of recCas9 may facilitate targeted agricultural breeding, or the study and treatment of human genetic diseases.

 
 
 
 
 
 

Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo 

John A Zuris, David B Thompson, Yilai Shu, John P Guilinger, Jeffrey L Bessen, Johnny H Hu, Morgan L Maeder, J Keith Joung, Zheng-Yi Chen, and David R Liu. Nature Biotechnology (2014), 33, 73-80.

ABSTRACT: Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. 

 
 
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This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells. 

 
 
 
 
 
 

Continuous evolution of site-specific recombinases with highly reprogrammed DNA specificities

Jeffrey L Bessen, David B Thompson, and David R Liu.

Poster presentation, 29th Annual Symposium of the Protein Society, Barcelona, 2015

ABSTRACT: The ability to precisely modify the genome of human cells has enormous potential as a novel therapy and a powerful research tool. In contrast to reprogrammable nucleases, such as TALENS or a Cas9/gRNA pair - which specifically cleave DNA but then rely on stochastic host cell processes to effect gene insertion - site specific recombinases directly catalyze genomic integration with high efficiency. A major limitation of this approach is that recombinases, such as Cre, natively bind with high specificity to long DNA target sequences (LoxP in the case of Cre) that do not exist in the human genome. Previous attempts at evolving Cre resulted in modest changes to its specificity, or required hundreds of rounds of manual protein evolution. We developed a Phage Assisted Continuous Evolution (PACE) selection for rapidly altering the DNA specificity of Cre recombinase towards a site present in a human genomic safe harbor locus. The PACE experiments resulted in Cre variants capable of recombining a substrate with nearly 50% of the nucleoties altered compared to LoxP. We successfully used one of these variants to integrate exogenous DNA into the genome of unmodified human cells. We are currently using sequencing methods to determine the specificity of the new recombinase clones.