Overcoming the AAV Size Limitation for CRISPR Delivery

Posted by Mary Gearing on Sep 16, 2020 9:00:00 AM


CRISPR and AAV: together they shall rule research!

Originally published Jul 14, 2015 and last updated Sep 16, 2020 by Beth Kenkel. 

CRISPR genome editing has quickly become a popular system for in vitro and germline genome editing, but in vivo gene editing approaches have been limited by problems with Cas9 delivery. Adeno-associated viral vectors (AAV) are commonly used for in vivo gene delivery due to their low immunogenicity and range of serotypes allowing preferential infection of certain tissues. However, packaging Streptococcus pyogenes (SpCas9) and a gRNA together (~4.2 kb) into an AAV vector is challenging due to its packaging capacity of AAV (~4.7 kb). While this approach has been proven feasible, it leaves little room for additional regulatory elements. Feng Zhang's group previously packaged Cas9 and multiple gRNAs into separate AAV vectors, increasing overall packaging capacity but necessitating purification and co-infection of two AAVs.

Cas9 orthologs: shorter, but just as potent and specific?

The two AAV strategies described above showed successful target modification, indicating that AAV is a good delivery vehicle for Cas9. However, to allow for more room for regulatory sequences while still fitting Cas9 and its gRNAs into one AAV, Cas9 must be made smaller. Previous attempts to “shrink” Cas9 include the use of St1Cas9 (~3.3 kb) from Streptococcus thermophilus and a rationally-designed truncated Cas9. Unfortunately, certain drawbacks limit the utility of these systems: St1Cas9 requires a very specific PAM sequence that limits the number of targetable loci, and truncated Cas9 has much lower efficiency than its wild-type counterpart. 

In search of a smaller, but equally potent Cas9 protein, the Zhang lab took a different approach. They analyzed over 600 Cas9 orthologs and found that they could be divided into two groups: longer orthologs approximately 1350 amino acids in size, which includes SpCas9, and shortern orthologs approximately 1000 amino acids in size. From the pool of shorter orthologs, only Staphylococcus aureus Cas9 (SaCas9, 1053 amino acids) displayed cleavage activity in mammalian cells. SaCas9 produced indels at a similar efficiency to SpCas9, leading the group to focus their efforts on SaCas9 characterization for in vivo studies.

One of the pitfalls of CRISPR/Cas9 genome editing is the potential for off-target effects. To compare the off-target effects of SpCas9 and SaCas9, Zhang’s group used an approach called BLESS (direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing). Using this sensitive method, they found that SaCas9 did not display higher levels of off-target activity than SpCas9, confirming its suitability for in vivo studies.

Testing AAV-SaCas9 in vivo

To test the efficiency of AAV-SaCas9 in vivo, the Zhang lab created an all-in-one SaCas9 and sgRNA construct using the liver-specific serotype AAV8. Since the efficiency of CRISPR/Cas9 genome editing varies across targets, they tested two genes in mice. For both genes, they saw indel formation and phenotypic changes as early as 1 week post-injection. Livers from these mice were histologically normal and liver injury markers were not increased compared to a control AAV-GFP. Not only did the AAV-SaCas9-sgRNA constructs mediate genome modification, but they did so without a substantial immune response or toxicity.

More AAV-based CRISPR systems

Smaller Cas proteins

SaCas9 isn't the only CRISPR enzyme that’s small enough to package into AAV.

  • At 984 amino acids in length, Cas9 from Campylobacter jejuni (CjCas9) is the smallest Cas9 ortholog characterized to date. Kim et al. successfully used CjCas9 with AAV to target genes in mouse muscle and eye tissue.
  • Neisseria meningitidis Cas9 (NmeCas9) at 1,082 amino acids, can also be packaged into AAV. NmeCas9 has the added advantage that it can be turned off by anti-CRISPR proteins. The Sontheimer lab used NmeCas9 with AAV to edit two different disease-causing loci in mouse liver
  • Two other smaller Cas proteins include Cas12b and CasX. While there are no papers yet published that deliver these Cas proteins with AAV, at just 1,108 and 986 amino acids, respectively, the size of both is within AAV’s packaging limit.
  • For CRISPR-based RNA editing, the REPAIR (RNA Editing for Programmable A to I Replacement), system is also small enough to deliver with AAV. This system fuses catalytically dead dCas13b to the catalytic domain of RNA deaminase ADAR2. Constructs containing the ADAR2 truncation ADAR2DD(delta984-1090) are approximately 4.1 kb in length, allowing them to be packaged in AAV.  

Split AAV approaches 

While finding smaller Cas9 orthologs works for some applications, it doesn’t eliminate the need to deliver larger cargo such as base editors and prime editors. While using AAV to deliver large transgenes might seem daunting, it’s actually a challenge that the field has overcome with split AAVs. In general split AAVs break a large transgene in two pieces and package each piece into an individual AAV. When a cell is transduced by both AAVs, the full length gene and/or protein is reconstituted.

One way to reconstitute a split protein like Cas9 is to use split inteins. Split inteins are a pair of naturally occuring polypeptides that, when at the ends of two proteins, mediates protein trans-splicing, similar to an intron in pre-mRNA splicing. In 2016, Fine et al developed a proof-of-concept split intein SpCas9 which had modest editing rates in HEK-293T cells when compared to the full-length SpCas9. In 2016, Chew et al. developed a split intein spCas9-AAV toolbox that retains the gene-targeting capabilities of full-length SpCas9. This set of plasmids includes AAV-Cas9C-VPR for targeted gene activation. Split inteins have also been used to express base editors.

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