Ph0ton t1_iy2ptbh wrote

It's a new technique that iterates upon existing work with integrases and fusion proteins in concert with CRISPR-Cas9 nickase to deliver huge packages of DNA. This existing work is still relatively new but is extremely promising, so PASTE has realized some of that potential. The pros are obvious: the ability to deliver large sections of DNA into multiple loci, dodging some of the deleterious effects of cellular repair pathways. As for cons, like many newer techniques, it requires expertise and development of various facets of the the insertion machinery. The promise of cas9 is any lab has the resources to develop a short guide RNA to make an edit, and they have a wealth of mature techniques to utilize said edit in most kinds of cells; also it's so easy a high schooler could run an interference assay (and they do).

As with any emerging tech, there will be unrealized challenges as it is deployed in various organisms, through numerous transfection techniques, but I wouldn't deign to speculate on those cons without a thorough review of the biochemistry (and other labs putting it into practice).


Ph0ton t1_iy1iimh wrote

Given that the fusion protein is usually characterized by chromosomal translocation, such abnormalities represent a huge restructuring of genomic material, and would not be a good target for a precision tool like CRISPR-Cas9. You could theoretically use something like CRISPR-Cas3 to shred the extraneous material, but to what end? I would think that such a cell is not worth repairing and should instead be targeted for destruction through other therapies.


Ph0ton t1_iy0nzod wrote

To be clear, CRISPR-Cas9 does not insert new genes. It's a nucleoprotein complex which simply creates a double-stranded break that allows for the opportunity for DNA to be inserted during the repair pathways. For a gene to be inserted, it must be first localized to the break site, then the right pathway must be initiated to insert the gene, and finally the whole sequence must be inserted to be functional.

In therapies where the patient's own cells are removed, edited, grown, and then transplanted back into the patient, the unlikelihood of a gene to be inserted correctly doesn't matter as much. With millions of cells, we only need a percentage to take up the gene, and then we only need to screen for those lucky few to culture for the transplantation. This is also complimented with cell culture techniques where we can arrest or cycle the cells in specific modes where it favors the better repair pathway. Delivery is also easier as we can use electroporation to insert (relatively) large payloads of genes or machinery. The same applies for edits within other cells; if we can culture them then it is trivial to "insert" a gene.

This is drastically more complex for editing in vivo, where we want a pre-existing population of cells to take up those inserts. For this challenge, we need other tools, such as CRISPR-Prime, PASTE, or different "flavors" of CRISPR-Cas proteins; from nature or designed ourselves. This is still a work in progress, and even delivering a gene of significant size is a challenge.

Generally speaking, the mechanics of the insert are extremely specific to the domain and objective of the research or therapy. If you are wondering about a specific development I'm happy to look at the paper to parse it for you.

Source: Former CRISPR-Cas3 researcher


Ph0ton t1_iw46mel wrote

Echoing this. CRISPR is a sea change but not a silver bullet. More developments in the vein of CRISPR-Prime will be great for developing models but besides cytotoxicity, currently there is an issue of cell-cycle arrest with most kinds of edits.

This will cause a survivorship bias among edited cells for those that can avoid that checkpoint and/or avoid cell death for critical, off-target effects.

Lots of work is being done though to minimize off-target edits, prevent cell-cycle arrest, and generally make CRISPR safer for therapy. With the millions of tools in nature, it's only a matter of time for us to find and perfect the right one that can make this viable.