A Match Made in Heaven: CRISPR and AAV

Posted by Mary Gearing on Jul 14, 2015 10:30:00 AM

CRISPR and AAV: together they shall rule research!

This post was updated on Dec 4, 2017.

CRISPR genome editing has quickly become the most 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 chimeric sgRNA together (~4.2 kb) into an AAV vector is challenging due to the low packaging capacity of AAV (~4.5 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 previous two AAV strategies described above showed successful target modification, indicating that AAV is a good delivery vehicle for Cas9. To maximize the genetic capacity of AAV, Gang Bao's group has developed a split-intein Cas9 that can be separated into two AAV cassettes, providing even more room for regulatory sequences and additional gRNAs in each cassette. However, to fit Cas9 and gRNAs into one AAV construct, the construct must be made even 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. 

Ran et al. recently developed a new strategy to overcome these drawbacks. To discover a shorter, but equally potent Cas9 enzyme, they analyzed over 600 Cas9 orthologs and found that they could be divided into two groups: one with orthologs of ~1350 amino acids, which includes SpCas9, and one with orthologs of ~1000 amino acids. From the pool of shorter orthologs, only Staphylococcus aureus Cas9 (SaCas9, 1053 aa) 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.

Designs used in CRISPR AAV VectorsOne 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, Ran et. al 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, Ran et al. 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 observed 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 AAV-SaCas9-sgRNA constructs mediate genome modification, but they did so without a substantial immune response or toxicity.

The work of Zhang’s group illustrates the potential of combining an advantageous vector delivery system (AAV) with a potent genome modification technique (CRISPR). In this “best of both worlds” scenario, in vivo genome editing without substantial toxicity or off-target effects will likely become much easier than we could have imagined.

If you’re interested in using SaCas9 in your research, the AAV targeting constructs are available from Addgene.

Other AAV-based CRISPR systems

SaCas9 isn't the only CRISPR enzyme to be successfully packaged 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.

In 2016, Chew et al. developed a split 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. 

REPAIR (RNA Editing for Programmable A to I Replacement), the new CRISPR-based RNA editing system, is also compatible with AAV delivery. 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.  


1. In vivo genome editing using Staphylococcus aureus Cas9. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F. Nature. 2015 Apr 9;520(7546);186-91. doi: 10.1038/nature14299. Epub 2015 Apr 1. PubMed

2. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, Sur M, Zhang F. Nat Biotechnol. 2015 Jan;33(1):102-6. doi: 10.1038/nbt.3055. Epub 2014 Oct 19. PubMed

3. Multiplex genome engineering using CRISPR/Cas systems. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Science. 2013 Feb 15;339(6121):819-23. doi: 10.1126/science.1231143. Epub 2013 Jan 3. PubMed

4. Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes. Fine EJAppleton CMWhite DEBrown MTDeshmukh HKemp MLBao G. Sci Rep. 2015 Jul 1;5: 10777. doi: 10.1038/srep10777. Pubmed.

5. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Kim E, Koo T, Park SW, Kim D, Kim K, Cho HY, Song DW, Lee KJ, Jung MH, Kim S, Kim JH, Kim JH, Kim JS. Nat Commun. 2017 Feb 21;8:14500. doi: 10.1038/ncomms14500. PubMed

6. A multifunctional AAV-CRISPR-Cas9 and its host response. Chew WL, Tabebordbar M, Cheng JK, Mali P, Wu EY, Ng AH, Zhu K, Wagers AJ, Church GM. Nat Methods. 2016 Oct;13(10):868-74. doi: 10.1038/nmeth.3993. Epub 2016 Sep 5. PubMed

7. RNA editing with CRISPR-Cas13. Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F. Science. 2017 Oct 25. pii: eaaq0180. doi: 10.1126/science.aaq0180.

8. CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox. Senís E, Fatouros C, Große S, Wiedtke E, Niopek D, Mueller AK, Börner K, Grimm D. Biotechnol J. 2014 Nov;9(11):1402-12. doi: 10.1002/biot.201400046. Epub 2014 Oct 6. PubMed

9. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Garneau JE, Dupuis MÈ, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadán AH, Moineau S. Nature. 2010 Nov 4;468(7320):67-71. doi: 10.1038/nature09523. PubMed

10. Crystal structure of Cas9 in complex with guide RNA and target DNA. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb 13. PubMed

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