Structures, mechanisms and engineering of CRISPR-Cas genome editors

Abstract
CRISPR-Cas systems harbor DNA nucleases that have emerged as powerful tools for precision genome editing in cells and organisms. The CRISPR-associated protein Cas9 is an RNA-guided nuclease that associates with an unusual dual-RNA guide structure and cleaves double-stranded DNA molecules complementary to a 20-nucleotide sequence in the guide RNA. Cas9 can be programmed using single-molecule guide RNAs to induce double-strand DNA breaks (DSBs) in genomic DNA, thereby enabling genome editing by exploiting DSB-activated DNA repair processes including non-homologous end joining and homology-directed repair.
Research in the Jinek laboratory focuses on understanding the molecular mechanisms of RNA-guided CRISPR-Cas genome editor nucleases. Our previous work has yielded atomic structures of Cas9 and its complexes with a guide RNA and a DNA target. These structures shed light on the molecular mechanism of Cas9-mediated DNA binding and cleavage and reveal the conformational transitions occurring during the process. Insights from our structural studies have enabled structure-guided engineering of the Cas9 nuclease to enhance its specificity and versatility, and to improve the efficiency of genome editing.
In addition to Cas9, our current studies have been focusing on complementary genome editing tools such as Cpf1/Cas12a, an RNA-guided nuclease that is capable of processing its own guide RNAs. We have determined crystal structures of Cas12a bound to a guide RNA and in a ternary complex with both an RNA guide and a full-length DNA target. These structures and corroborating biochemical experiments reveal the catalytic mechanism of guide RNA processing and the structural basis for seed-sequence dependent DNA targeting.
Overall, these studies have advanced our understanding of CRISPR-Cas systems and provide the structural framework for their ongoing development towards a new generation of genome editing tools and technologies.

 

Biosketch
Martin Jinek is an Assistant Professor in the Department of Biochemistry at the University of Zurich. His research focuses explores two main topics – (i) RNA biology and (ii) CRISPR-Cas systems and their use as a genome editing technology. Originally from the Czech Republic, Martin Jinek studied Natural Sciences at the University of Cambridge (UK). In 2006, he received his PhD from the European Molecular Biology Laboratory (EMBL) in Heidelberg (Germany) where he conducted his doctoral research in the lab of Prof. Elena Conti. He then moved to the University of California in Berkeley for postdoctoral research with Prof. Jennifer Doudna, where his work led to the discovery of the biochemical function of the RNA-guided endonuclease Cas9 and made fundamental contributions towards developing the CRISPR-Cas9 genome editing technology. Since starting his independent research group at the University of Zurich in 2013, Martin Jinek has studied the molecular mechanisms of CRISPR-Cas genome editor nucleases in atomic detail. In recognition of his work, he has received several awards, including an ERC Starting Grant (2013), the EMBL John Kendrew Young Scientist Award (2014) and the Friedrich Miescher Award of the Swiss Society for Molecular and Cellular Biosciences (2015). He is an EMBO Young Investigator and in 2017 became an International Research Scholar of the Howard Hughes Medical Institute.