Developing Prime Editing in Organoids as a Modeling Tool and Treatment Strategy

Review of “Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids” from Life Science Alliance by Stuart P. Atkinson

The error-prone repair of DNA double-stranded breaks and off-target effects associated with CRISPR/Cas9-mediated genome editing [1-4] have promoted the development of base editors [5] and prime editing [6], which support gene-editing without the need to generate DNA insults. Base editors represent an efficient and specific means of generating mutations [7]; however, their scope is limited to particular types of mutations. As an alternative, prime editing has the potential to repair an estimated 89% of disease-causing variants [6].

In a recent study published in Life Science Alliance, researchers led by Hans Clevers (Hubrecht Institute, Utrecht, the Netherlands) describe their application of prime editing (which comprises a nickase-cas9 fused to a reverse transcriptase that synthesizes the edit at the target site) in human organoids to introduce cancer-related mutations and then repair cystic fibrosis-associated mutations to the fibrosis transmembrane conductance regulator (CFTR) gene [8]. Their organoid-based findings provide evidence for the immense potential of prime editing in the realms of disease modeling and treatment but highlight the need for further innovation.

Geurts et al. discovered that prime editing supported the modeling of oncogenic mutations (ten target sequences for TP53 and APC) in two organoid models; however, they also highlighted varying editing efficiencies. Four targets resulted in the accurate modeling of mutations with a 25% efficiency in human adult stem cell-derived colonic organoids and a 97% efficiency in hepatocyte organoids, suggesting that prime-editing efficiencies will differ significantly between organoid tissue types. Unfortunately, the authors also discovered some undesired outcomes at the target sites.

After providing evidence for the validity of the concept, the authors next employed prime editing to repair a cystic fibrosis-associated mutation in the CFTR gene in colonic organoids. While prime editing failed to induce any detectable off-target effects (thereby providing evidence for this strategy as a safe approach to gene repair), the authors noted low efficiency and the presence of undesired mutations at the target sites, including indel formation.

While these findings provide evidence for the immense potential of prime editing, the authors note the need for further platform optimization to decrease unintended target site modifications to allow the widespread implementation of this approach in disease modeling and therapeutic gene repair.

For more on organoids, prime-editing, disease modeling, and novel treatments for cystic fibrosis, stay tuned to the Stem Cells Portal!

References

1. Fu Y, Foden JA, Khayter C, et al., High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology 2013;31:822-826.
2. Pattanayak V, Lin S, Guilinger JP, et al., High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nature Biotechnology 2013;31:839-843.
3. Cho SW, Kim S, Kim Y, et al., Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Research 2014;24:132-141.
4. Kosicki M, Tomberg K, and Bradley A, Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nature Biotechnology 2018;36:765-771.
5. Gaudelli NM, Komor AC, Rees HA, et al., Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 2017;551:464-471.
6. Anzalone AV, Randolph PB, Davis JR, et al., Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 2019;576:149-157.
7. Geurts MH, de Poel E, Amatngalim GD, et al., CRISPR-Based Adenine Editors Correct Nonsense Mutations in a Cystic Fibrosis Organoid Biobank. Cell Stem Cell 2020;26:503-510.e7.
8. Geurts MH, de Poel E, Pleguezuelos-Manzano C, et al., Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids. Life Science Alliance 2021;4:e202000940.