A new part for gene scissors
New genome editing tools are described
Alexandra Medvedev, Indicator
Researchers from the Skolkovo Institute of Science and Technology, Peter the Great St. Petersburg Polytechnic University and the Institute of Gene Biology of the Russian Academy of Sciences together with colleagues from the USA described two new compact Cas9 nucleases used by the CRISPR/Cas system for DNA cutting. These proteins can add to the set of tools for genome editing, scientists have shown that one of the nucleases can work in human cells, and this opens up prospects for its use in biomedical applications. The results of the study are published in the journal Nuclear Acids Research (Fedorova et al., PpCas9 from Pasteurella pneumotropica – a compact Type II-C Cas9 ortholog active in human cells).
CRISPR/Cas is a system of adaptive immunity of bacteria and archaea, borrowed into genomic editing technology. Cas nucleases are capable of using CRISPR RNA to cut DNA sequences in certain places. In studies, as a rule, Cas9 nucleases of the SpCas9 type from streptococcal bacteria are used, they are effective and relatively simple. To recognize targets, SpCas9 requires a fairly short PAM sequence (PAM, protospacer adjacent motif - motif adjacent to the protospacer) of several DNA nucleotides necessary for successful genome cutting. The disadvantage is that this large protein may not fit into the particle of the adenoassociated virus (AAV), which delivers the system inside the cell.
The authors of the article described two new small nucleases Cas9, DfCas9, obtained from the bacterium Defluviimonas sp.20V17, living in hydrothermal vents, and PpCas9 – from the bacterium Pasteurella pneumotropica, a mammalian parasite. These nucleases simultaneously have a size small enough for delivery to AAV and recognize relatively short DNA sequences. The new nucleases belong to the CRISPR/Cas type II-C systems, their structure is very similar to that of SpCas9, but at the same time these proteins have a number of unique features.
"Yes, Cas9 type II-C effectors, as a rule, require longer PAM sequences, but this is only an observation based on the study of a limited number of II-C nucleases described to date. For example, in bacteria Staphylococcus auricularis recently discovered the protein SauriCas9, which similarly to the protein PpCas9 requires a short PAM sequence – just two “letters". I think other Cas9 type II-C enzymes requiring short PAM sequences will be discovered soon. Small-sized Cas9s with different PAM requirements increase the number of potential DNA targets in the genomes of eukaryotes and prokaryotes that can be edited by CRISPR/Cas systems," notes co–author Yana Fedorova.
Experiments have shown the effectiveness of two new nucleases, and PpCas9 has been found to be active in human cells. To evaluate the effectiveness of PpCas9 in eukaryotic cells, additional studies will be required, however, according to the authors, this protein can be used along with traditional nucleases that are currently used in microbial biotechnologies and biomedicine for genome editing, and expand the use of CRISPR/Cas systems.
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