At a cellular level, organisms face two fundamental challenges: maintaining integrity of the genome in response to mobile genetic elements and mutagens, and expressing a specific repertoire of genes at the correct time and proper level. It is now widely recognized that noncoding RNAs play crucial and surprisingly diverse roles in controlling both the expression of DNA, via transcriptional and posttranscriptional gene regulation, and the content of DNA itself, by mediating sequence-specific DNA cleavage events. The recent discovery of pervasive genome defense systems in bacteria and archaea known as CRISPR–Cas (Clustered Regularly Interspaced Short Palindromic Repeats–CRISPR-associated), and the development of these systems for genome engineering, highlight the biological power and technological potential of RNA-guided DNA control.
The Sternberg Lab broadly strives to expand our understanding of the ways in which noncoding RNAs conspire with effector proteins to target DNA. Focusing on evolutionarily distinct but analogous systems in both prokaryotes and eukaryotes, and using a combination of biochemistry, structural biology, biophysics, and genetics, we are uncovering new biological function while simultaneously advancing novel tools with which to precisely manipulate the genome.
CRISPR-CAS IMMUNE SYSTEM DIVERISTY
CRISPR–Cas systems are remarkably diverse, yet much of the research has focused on Cas9-containing Type II systems. We showed that many of the same strategies that Cas9 uses during target interrogation are similarly exploited by Type I systems, and we continue to explore other immune system subtypes and their potential for genome engineering applications.
DNA TARGET SEARCH AND RECOGNITION
How do CRISPR RNA-guided proteins hunt down complementary DNA target sequences within the vast expanse of the genome? Using single-molecule, biochemical, and high-throughput approaches, we showed that the Cas9 endonuclease is specifically recruited to genomic hotspots and interrogates DNA via directional DNA unwinding. Ongoing work is aimed at investigating target search for other CRISPR–Cas effectors.
RNA-GUIDED DNA INTEGRATION
We recently discovered that a class of bacterials transposons encoding their own CRISPR–Cas systems can mobilize using RNA-guided DNA integration. This upends the existing paradigm of CRISPR–Cas systems existing only for defense against mobile genetic elements, and furthermore lays the foundation for a new genome engineering technology that obviates the requirement for DSBs and homology-directed repair.
CONFORMATIONAL CONTROL OF DNA CLEAVAGE
Early genome engineering experiments demonstrated that DNA binding by Cas9 is far more promiscuous than DNA cleavage. To understand the mechanistic basis, we used fluorescence techniques to reveal that nuclease domain activation is carefully regulated by conformational dynamics. More recently, we harnessed this knowledge to build higher-fidelity genome editors.