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 biochemical, structural, and biophysical approaches, we are uncovering new biological function while simultaneously advancing novel tools with which to precisely manipulate the genome.
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.
CRISPR TOOL DEVELOPMENT
We are continually interested in harnessing mechanistic knowledge about CRISPR–Cas function to build new tools. Towards this end, we used structure-guided protein engineering to develop one of the first split-Cas9 enzymes, and to develop higher-fidelity Cas9 variants for genome editing. We also showed that CRISPR–Cas9 could function as a programmable system for RNA targeting applications.
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.
CRISPR-CAS Immune SYstem Diversity
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.