CRISPR / Cas9 and AAV

Posted by Wendy Wilson on March 28th, 2017

CRISPR / Cas9 has become the most popular system for in vitro genome editing, but the in vivo gene editing method is still limited by the Cas9 import problem. The adeno-associated virus vector (AAV) is often used for in vivo introduction of genes due to low immunogenicity. However, Streptococcus pyogenes (spCas9) and chimeric sgRNAs (4.2 kb in total) are quite challenging to be packaged into the adenoviral vector because the AAV can only accommodate up to 4.5 kb. Although this approach has proven to be feasible, the locations of other regulatory elements are not much.

In order to maximize the ability of AAV, a peptide-containing Cas9 was divided into two AAV expression frames, leaving more space for regulatory sequences and gRNAs. However, in order for Cas9 and gRNA to be together, this construct must be smaller. Previous attempts were to use St1Cas9 (~ 3.3 kb) from Streptococcus thermophilus and truncated Cas9. They had some drawbacks: St1Cas9 required a very specific PAM sequence that limited the number of targetable loci, while truncated Cas9 was much less efficient.

In order to find shorter, but equally effective Cas9 enzymes, more than 600 Cas9 orthologues were analyzed. One class was about 1,350 amino acids, including SpCas9, while the other had about 1000 amino acids. Of those shorter homologs, only Staphylococcus aureus Cas9 (SaCas9) showed cleavage activity in mammalian cells. SaCas9 produced insertions / deletions, and its efficiency was similar to SpCas9, which made the research team focus on SaCas9 research.

Another drawback of the CRISPR / Cas9 genome editing is the possibility of off-target effects. In order to compare the off-target effects of SpCas9 and SaCas9, the researchers used a method called BLESS. Using this sensitive approach, they found that SaCas9 did not show a higher off-target activity than SpCas9, confirming that it was suitable for in vivo studies.

Because CRISPR / Cas9 gene editing efficiency varies with the target, the researchers tested two genes in mice. For both genes, they observed changes in insertions and deletions and phenotypic changes after one week of injection. Mice liver was normal in histology, and no significant increase in liver injury markers compared to GFP-AAV control.
The new Cas9 expands the Cas9 list to help us build a better disease model, identify out some mechanisms, and develop new therapies.

Like it? Share it!


Wendy Wilson

About the Author

Wendy Wilson
Joined: March 28th, 2017
Articles Posted: 1