Newly identified Cas9 nucleases can improve genome editing

Scientists from Skolkovo Institute of Science and Technology (SKOLTECH) and their collaborators from Russia and the United States have explained two new, compact Cas9 nucleases—the cutting constituents of CRISPR-Cas systems—that will possibly extend the Cas9 toolbox for genome editing.

Cas9Image Credit: vchal /

Among these two nucleases, one has been demonstrated to function in human cells and therefore can have biomedical applications. The article was published in the Nucleic Acids Research journal.

The genome editing technology, CRISPR-Cas, was borrowed from bacteria and depends on Cas nucleases; when directed by CRISPR RNAs, these enzymes can degrade the sequences of target genes—they are the blades in the Nobel Prize-worthy “genetic scissors.”

Streptococcus pyogenes one, Type II-A SpCas9, is the most popular Cas9 nuclease in research applications. It is both efficient and comparatively simple, since one huge protein attaches to crRNA and also cleaves the DNA; it also needs a short PAM—a nucleotide sequence bookending the site of interest used by enzymes to detect and “read” it.

However, SpCas9 is a massive protein that creates an issue when one wants to, for example, when using an adeno-associated viral (AAV) particle as a vehicle to deliver the “genetic scissors” into a cell.

Preferably, one would wish to fit the gene encoding the Cas protein and also the sequences for guide RNAs within a single particle, and that size limit needs shorter types of Cas9 nucleases. But those shorter nucleases also need longer and more complex PAMs; therefore, scientists faced a tradeoff between choice of targets and protein size.

In their latest study, Iana Fedorova, who lately defended her Skoltech PhD, and Aleksandra Vasileva, a research scientist from the Skoltech Severinov laboratory, and their collaborators explained two new small Cas9 nucleases obtained from Pasteurella pneumotropica, a common bacterium present in rodents and other mammals, and Defluviimonas sp.20V17, a bacterium dwelling in hydrothermal vents.

Such nucleases happen to be sufficiently small for an AAV vector and have comparatively short PAMs—a “best of both worlds” option when it comes to Cas9 enzymes.

The novel nucleases are associated with Type II-C CRISPR-Cas systems, which are generally represented by smaller Cas9 effectors when compared to SpCas9. Such proteins assume a conservable bilobed architecture analogous to other Cas9 proteins, but also exhibit special features—that is, they do not contain many insertion subdomains and possess a smaller Wedge domain (the domain that is responsible for communication with single-guide RNA scaffold)—and, therefore, are more compact.

Indeed, type II-C effectors tend to require longer PAM sequences, but it is just an observation based on a limited number of Type II-C effectors characterized to date. For example, the recently found SauriCas9 from Staphylococcus auricularis, similarly to PpCas9, requires a short PAM (5'-NNGG-3').”

Iana Fedorova, PhD, Skolkovo Institute of Science and Technology

Fedorova continued, “And most likely more Type II-C Cas9 enzymes requiring short PAMs will be found soon. These small Cas9 proteins with different PAM requirements expand the number of potentially editable DNA targets in eukaryotic and prokaryotic genomes.”

Experiments and in vitro studies performed in bacteria demonstrated that both these nucleases can efficiently cleave DNA, and the P. pneumotropica Cas9 nuclease is active in human cells. It also turned out to be quite analogous to other Cas9 nucleases, which had been demonstrated to work in eukaryotic cells—Nme1Cas9 and Nme2Cas9.

While more studies are required to establish the efficiency of these nucleases, the authors are hopeful that they may provide a feasible option to the more traditional nucleases employed in biomedical genome editing and microbial engineering.

Fedorova noted that initial research of PpCas9 off-targeting (unintentional modifications) has demonstrated that this specific enzyme has fair specificity. However, to prove that PpCas9 is sufficiently specific to be regarded as a genome editing tool, further studies using more advanced techniques are required.

Moreover, it looks like PpCas9 demonstrates selectivity in targeting of different genes in cells. It may reduce the range of possible PpCas9 genomic targets, and the nature of this bias is a subject of further studies.”

Iana Fedorova, PhD, Skolkovo Institute of Science and Technology

Journal reference:

Fedorova, I., et al. (2020) PpCas9 from Pasteurella pneumotropica — a compact Type II-C Cas9 ortholog active in human cells. Nucleic Acids Research.


The opinions expressed here are the views of the writer and do not necessarily reflect the views and opinions of AZoLifeSciences.
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