NEW YORK (GenomeWeb) – A new study by researchers at the University of Utah School of Medicine has found that the architecture of DNA — specifically the presence or absence of nucleosomes — can affect the efficiency of genome editing by CRISPR-Cas9 in vivo.
In their study, published yesterday in the Proceedings of the National Academy of Sciences, the researchers, led by Dana Carroll at the university's Department of Biochemistry, wrote that editing targets bound by nucleosomes — segments of DNA wound around a core of eight histone proteins— are cut much less efficiently than targets that are not part of nucleosomes, or had them depleted. This could help inform target selection in order to improve the efficiency of editing in vivo, the team noted.
The researchers also said that the presence of nucleosomes does not inhibit editing with zinc-finger nucleases (ZFNs), which cleave equally well at nucleosome-occupied and nucleosome-depleted sites. This could also have implications for the selection of editing technology, both for research and clinical purposes, the investigators added.
Nucleosome locations at the yeast HO promoter and their dynamics have been well characterized in previous research. To explore the effect of chromatin on Cas9 cleavage in this study, the researchers designed single-guide RNAs to DNA sequences in the constitutive nucleosome-depleted region (NDR) about 1,300 base pairs upstream of the translation start and to multiple nucleosome-bound sites. They assessed cleavage after various times of sgRNA induction and found that a target in the NDR 1,339 base pairs upstream of the translation start was cut much more efficiently than one in the nucleosome-bound site 184 base pairs upstream.
In another experiment, two targets in the NDR at 1,363 and 1,339 base pairs upstream were effectively cleaved, while four nucleosome-bound sites at 1,670, 1,235, 689, and 184 base pairs upstream showed weak or no cleavage. This supported the suggestion that nucleosomes inhibit Cas9 cleavage in vivo, the authors said.
Experiments with ZFNs, however, showed that nucleosomes did not inhibit editing efficiency. This indicated to the researchers that ZFNs are largely impervious to nucleosomes on their target.
"The interference by nucleosomes with Cas9 cleavage that we document is quite striking," the authors wrote. "Overall, every manipulation of occupancy had the effect expected for nucleosome inhibition of Cas9 cleavage. What impact will our findings have on experimental genome editing? Chromatin structure will be more important in some contexts than others, and there will certainly be differences in the inherent effectiveness of different guide RNAs. In many situations, finding a good Cas9 target and producing an effective guide RNA is simply a matter of testing a modest number of candidates."
The investigators noted that knowledge of nucleosome locations would likely be most important for choosing editing targets in certain situations, such as making animal models of specific human genetic diseases, and in certain human therapeutic applications.
Importantly, they added, some Cas9 variants may be more or less susceptible to nucleosome inhibition than the Streptococcus pyogenes enzyme used in their study, and it seems likely that there will be effects on more distant relatives like Cas12a and Cas12b.
"While the CRISPR platform was substantially inhibited by nucleosomes in our experiments, ZFNs were not. In its natural bacterial setting, Cas9 would not encounter nucleosomes, so there would be no pressure to acquire that capability," they wrote. "Obviously, this is not a significant impediment to general use of Cas9 in eukaryotic cells.... Zinc fingers, in contrast, are found in large families of natural eukaryotic transcription factors, and they have evolved to function in a chromatin context …. Knowing that nucleosomes interfere directly with Cas9 cleavage, but not with ZFN cleavage, is useful information. It may explain some cases of unanticipated sgRNA failure, and it should guide specific applications where precisely located, high-efficiency cleavage is necessary."