The AAVantages of AAV in CRISPR Screens

Forward genetics screens are a valuable part of the molecular biology toolbox to identify new target genes for drug discovery or to understand the intricacies of molecular pathways. These screens have gotten larger, easier, and more comprehensive thanks to the consistent development and improvement of CRISPR-based technologies over the past decade. CRISPR screens are easily customizable and enable the targeting of hundreds to tens of thousands of genes with minimal effort. The versatility of Cas effectors allows for a wide variety of screens, including interference, activation, and knockout.

Most CRISPR screens rely on lentiviral systems, a fact supported by the wide array of lentiviral-based CRISPR libraries available at Addgene. The large packaging capacity and stable expression of lentiviral vectors make them an attractive option. However, safety requirements and expression limitations may be too constraining for some experiments, particularly in vivo ones. In those cases, AAV vector screens may be AAVantageous!

In vivo, in vitro, ex vivo

CRISPR screens can be carried out using a few different methods — in vivo, in vitro, and ex vivo. The general process of these screens is the same:

1. Deliver CRISPR library to cells of interest.
2. Select for cells of interest (positive or negative selection).
3. Next-generation sequencing (NGS) to identify genes of interest.

Figure 1: Overview of in vitro AAV CRISPR screens. Created with BioRender.com.

The in vitro option requires a bit less work for the first two steps, as no mice are involved. Ex vivo starts with transducing cells with the CRISPR library in vitro before transplanting them into mice. For in vivo screens, mice are directly infected with the CRISPR library. In vivo screens can become costly, so having an efficient and effective delivery method is vital for the best outcomes.

The go-to in vivo option

AAV has become the preferred delivery method for in vivo CRISPR screens. AAV vectors can be made at high titers equal to those achieved with lentivirus but offer an improved safety profile, customization, and enhanced expression in many mouse tissues.

Safety

AAV and lentivirus have different safety profiles that often affect which is chosen for certain experiments. AAV can be safely handled at BSL-1, while lentivirus requires a BSL-2 setup. This requires a less involved setup when working with mice and slightly less red tape for approval processes. Additionally, while both AAV and lentivirus have low immunogenicity, AAV still beats lentivirus. In mice, AAV is almost entirely non-immunogenic — meaning when conducting in vivo CRISPR screens, AAV is unlikely to cause bad reactions in mice.

Customization

AAV offers a level of customization that is unmatched by other viral vector delivery methods. Lentivirus has limited packaging options, with most lentiviral vectors being packaged using VSV-G. In contrast, AAV can be packaged in a variety of capsids. To date, at least 12 different capsids, or serotypes, have been identified in nature, while many more have been engineered in labs. This vastly increases the possibilities when designing screens. Each serotype has its own tropism, or a preference to infect specific cell or tissue types. Using serotypes that have a known tropism for your cells of interest — for example, AAV8 for kidney cells — can help improve infection rates in your experiment.

Figure 2: Visual representation of common AAV serotypes used for different tissues. Created with BioRender.com.

Beyond the identified serotypes, AAV also has psuedotypes. Psuedotyping is the process of making a hybrid AAV, with the genome from one serotype and the capsid of another. Researchers have used these psuedotypes to further improve the tropism of AAV vectors, enhancing their affinity for specific cell and tissue types.

The combination of serotypes and psuedotypes means you can customize your CRISPR screen to a high degree. This is particularly helpful when investigating tissue-specific diseases, as you can target your library to a significant degree using different types of AAV.

Expression

Another advantage of AAV vectors is how the cargo — in this case, the CRISPR components — are expressed. Unlike lentivirus, cargo delivered by AAV does not integrate into the host genome. AAV instead relies mainly on episomal expression. Episomes are circular pieces of DNA that reside outside a host cell’s genome. In this state, AAV cargo can be stably expressed in non-dividing cells for long periods of time. This reduces the chance of integration at random locations that could cause deleterious effects.

It is important to note, however, that while AAV rarely integrates into the genome, CRISPR may increase this rate. Specifically, portions of the AAV genome can integrate at double-stranded break points caused by CRISPR-Cas9 editing. The good news is that this integration is not random, nor is it a global occurrence, as integration sites seem to only occur in the target cells or tissues.

AAV also shows improved transduction efficiency over lentivirus in many mouse tissues. This will help improve your selection pool for downstream NGS and means you can use smaller volumes of your library for each experiment.

Additional considerations

One of the downsides to using AAV is the limited packaging capacity. Lentiviruses have capacities of 8–12 kb, while AAV only has approximately 4.7 kb — which means that when using Cas9, you’re already almost out of space! Don’t let this get you down for too long, though, because many solutions have been found to get around this restriction.

The most obvious workaround is to use a multi-plasmid system, where you have one AAV vector dedicated to expressing Cas9, while the other houses the gRNA and scaffold. If you don’t feel like doubling the amount of cloning, another option is to use variants of Cas9, like split Cas9, or a smaller Cas9 ortholog like SaCas9. You could even venture away from the comfort of Cas9, and opt for a completely different Cas effector, like Cas12a (Cpf1). Even more creative options involve generating strains of mice that stably express Cas effectors in specific tissues, eliminating the need to deliver the Cas protein altogether.

Platforming AAV

Interest in interrogating and mapping the genetic landscape of tissue-specific diseases has grown exponentially, aided by advancements in in vivo CRISPR screens. AAV vectors have played a large role in these advancements, due to their many benefits for in vivo models. Over the past few years, AAV CRISPR screens have been used to map tumor suppressors in mouse liver, interrogate immune cells to improve immunotherapy in glioblastoma, and generate programmable screening platforms for mouse tissues. Your AAVancement could be next!

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References and Resources

References

Braun, C. J., Adames, A. C., Saur, D., & Rad, R. (2022). Tutorial: design and execution of CRISPR in vivo screens. Nature Protocols, 17(9), 1903–1925. https://doi.org/10.1038/s41596-022-00700-y

Hanlon, K. S., Kleinstiver, B. P., Garcia, S. P., Zaborowski, M. P., Volak, A., Spirig, S. E., Muller, A., Sousa, A. A., Tsai, S. Q., Bengtsson, N. E., Lööv, C., Ingelsson, M., Chamberlain, J. S., Corey, D. P., Aryee, M. J., Joung, J. K., Breakefield, X. O., Maguire, C. A., & György, B. (2019). High levels of AAV vector integration into CRISPR-induced DNA breaks. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-12449-2

Pupo, A., Fernández, A., Low, S. H., François, A., Suárez-Amarán, L., & Samulski, R. J. (2022). AAV vectors: The Rubik’s cube of human gene therapy. Molecular Therapy, 30(12), 3515–3541. https://doi.org/10.1016/j.ymthe.2022.09.015

Ramani, B., Rose, I. V., Teyssier, N., Pan, A., Danner-Bocks, S., Sanghal, T., Yadanar, L., Tian, R., Ma, K., Palop, J. J., & Kampmann, M. (2023). CRISPR screening by AAV episome-sequencing (CrAAVe-seq) is a highly scalable cell type-specificin vivoscreening platform. bioRxiv (Cold Spring Harbor Laboratory). https://doi.org/10.1101/2023.06.13.544831

Santinha, A. J., Klingler, E., Kuhn, M., Farouni, R., Lagler, S., Kalamakis, G., Lischetti, U., Jabaudon, D., & Platt, R. J. (2023). Transcriptional linkage analysis with in vivo AAV-Perturb-seq. Nature, 622(7982), 367–375. https://doi.org/10.1038/s41586-023-06570-y

Wang, J., Gessler, D. J., Zhan, W., Gallagher, T. L., & Gao, G. (2024). Adeno-associated virus as a delivery vector for gene therapy of human diseases. Signal Transduction and Targeted Therapy, 9(1). https://doi.org/10.1038/s41392-024-01780-w

Additional resources on the Addgene Blog

• Overcoming the AAV Size Limitation for CRISPR Delivery
• Genome-wide Screening Using CRISPR
• Performing In Vivo CRISPR Screens Using the FITS Approach

Additional resources at addgene.org

• CRISPR pooled libraries
• Guide to pooled libraries
• CRISPR guide
• AAV guide