Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

摘要

CRISPR is a method of editing the genetic material inside living cells and has enabled dramatic advances in a broad variety of research fields in recent years. The method relies on a bacterial enzyme called Cas9 that can be programmed, via short guide molecules made from RNA, to target specific sites in the cell’s DNA. Once bound to its target, the Cas9 enzyme cuts the DNA molecule; this often leads to changes in the DNA sequence. In nature, bacteria use the CRISPR-Cas9 system to defend themselves against viruses. However, this system also works in other cell types and can be reprogrammed to target almost any site in the DNA. To date, the CRISPR-Cas9 system has been used in fungi, worms, flies, plants, mammals and other eukaryotes. Yet, unlike in bacteria, much of the DNA in eukaryotes is wrapped around proteins called histones to form units referred to as nucleosomes. This means eukaryotic DNA is often tightly packaged, which makes it less accessible to other proteins. Nevertheless, eukaryotic DNA will spontaneously detach and reattach to the histones – a phenomenon that is commonly known as DNA “breathing”. Also, protein machines known as chromatin remodelers can move, assemble and take apart the nucleosomes in eukaryotic cells. However, because there is much still to learn about how CRISPR-Cas9 works in eukaryotic cells, it is not clear how nucleosomes affect this system’s activity. Isaac et al. have now used a simplified biochemical system to test how nucleosomes and chromatin remodelers affect CRISP-Cas9 activity. The system comprised purified Cas9 enzymes, short guide RNA molecules and nucleosomes. The experiments revealed that the Cas9 enzyme was able to cut DNA on nucleosomes when the DNA sequence allowed more spontaneous breathing or when chromatin remodelers were present to destabilize or move the nucleosome out of the way. These results suggest that by taking the placement of the nucleosomes into account, researchers can better predict how effective the CRISPR-Cas9 system will be at targeting a specific DNA sequence in a eukaryotic cell. The findings also suggest ways to make genome editing with CRISPR-Cas9 even more efficient.