Crispr can do multiple things. If you want to shutoff a mutant gene, crispr cuts DNA that introduces mutations that eventually turn the gene off. Yes, you can also cut DNA and paste in a gene sequence with Crispr to fix faulty genes that you can’t just shutoff. There are also twists like Crispr base editors that can fix a single mutation without the need to cut DNA that causes a double strand break.

Contrast that to AAV. AAV doesn’t really integrate into a genome (well isn’t supposed to in theory) - they work by creating what’s known as an episome (i.e a circular piece of dna that persists in cells and gets translated into the desired protein). AAVs can only shutoff a mutant gene if they carry a payload like siRNA/microRNA or something. AAVs never really fix the mutant gene, the episome just expresses the protein that’s not working. I suppose over the long run AAVs might not really ‘cure’ a genetic disease, because the episome will likely dilute out over time with cell divisions. You can only really administer an AAV once too because of immunogenicity issues.

Also, you could use AAVs to deliver genes that encode for Crispr, so it isn’t like they’re mutually exclusive. There are pros and cons of using either of these approaches for a gene therapy. It depends on your strategy, target population, and overall risk.

There is actually a surprisingly large overlap between two systems. Both CRISPR and AAV can be used for targeted integrations (by using site specific homologous regions to direct the HDR machinery). AAV can also be used for a random integrations and expression from long-term episomes.

The major differences between the methods are some of the more practical aspects rather than the actual repair mechanism; things like delivery methods, necessary components, penetrance in in vivo systems, etc.

Delivery/components: CRISPR requires the transfection or injection of multiple components (Cas Proteins, sgRNAs, tracrRNA depending on the organism, homologous repair templates, etc). AAV requires transfection of a multi-component virus which encodes all necessary machinery. Additionally CRISPR generally requires introduction into either a germ or progenitor cells so that the edit can be spread throughout the entire organism, or into a subset of cells (which is easy to do when they are in culture but nearly impossible to do in a living organism (yet!)). AAV is much easier to target a particular subset of cells to make edits (such as specifically transfecting lung tissue or organ; see gene therapy methods for treating cystic fibrosis in lung tissue).

Penetrance: See above. If either methods are used to edit germ cells, then the edits will be organism wide in the mature adult (ideally). On the flip side, mosaicism is nearly always unavoidable using CRISPR when targeting somatic cells in a living organism. There are some exceptions (see CarT therapy) where certain cell populations can be removed from the body, edited and returned. However for most cell populations there is no feasible way to edit them specifically and efficiently. There is also a chance of mosaic integrations with AAV but without examining particular cases it is difficult to say what the percentages would be.

Overall: CRISPR and AAV are comparable when editing germ cells. AAV methods are much better when targeting somatic cells in a living system such a patient require gene therapy. Hope this helps. Source: Post-doctoral researchers who utilizes CRIPSR frequently in my own research (specifically in the nematode C. elegans), and has dabbled in AAV methods in cell culture.

Linking AAV article for more specifics:

"The Role of Recombinant AAV in Precise Genome Editing" doi: 10.3389/fgeed.2021.799722 PMID: 35098210

In actual use, AAVs often cannot integrate into the cellular genome. It's just used to temporarily deliver something else. In fact it's a commonly used vector for CRISPR/Cas systems.

But it's true that unmodified AAVs are capable of integration. It's a lot more specific about that than lentiviral vectors, because AAVs mostly just integrate into the human genome at the AAVS1 site.

Lentiviral vectors can insert at many, many sites throughout the genome. The same applies for transposon systems such as Sleeping Beauty and PiggyBac.

As for CRISPR/Cas, that's a whole range of systems that is mainly distinguished by the ability to easily program targeting of a specific sequence (in almost any context). That can be used to insert something at a specific genomic site, but it can also be used for all sorts of other things. Some Cas enzymes don't even target DNA but instead target RNA.

> > > > > Contrast that to AAV. AAV doesn’t really integrate into a genome (well isn’t supposed to in theory) - they work by creating what’s known as an episome (i.e a circular piece of dna that persists in cells and gets translated into the desired protein). AAVs can only shutoff a mutant gene if they carry a payload like siRNA/microRNA or something. AAVs never really fix the mutant gene, the episome just expresses the protein that’s not working. I suppose over the long run AAVs might not really ‘cure’ a genetic disease, because the episome will likely dilute out over time with cell divisions. You can only really administer an AAV once too because of immunogenicity issues.

That's often how they are used in practice, but unmodified AAVs are capable of insertion, in humans mostly at the AAVS1 locus.

Yes, that’s why I wrote “in theory”. In reality, it isn’t that clean, even with modification.

Integration may not just be occurring at AAVS1, but all over the place. Studies have detected things like complex vector rearrangements and truncated vector genomes across multiple animals models inserted around transcriptional units. There seems to be no preference for gene coding regions and no clustering of integration sites.

In fact, The FDA had a CTGTAC meeting in 2021 to discuss these issues: https://www.fda.gov/advisory-committees/cellular-tissue-and-gene-therapies-advisory-committee/2021-meeting-materials-cellular-tissue-and-gene-therapies-advisory-committee

Multiple ways of gene editing have existed for a while. CRISPR is way more scalable. Other forms of genome editing are either more expensive to target a gene of interest. Other technologies are imprecise. We've been able to insert a gene into a genomes for decades, but it also just randomly inserts it all over the genome.