Exploring the auxin-inducible degron system in CRISPR-engineered mammalian models

Abstract

The condensin complexes are highly conserved proteins that are essential for the proper and accurate partitioning of genetic material across all domains of life. In mammalian organisms, the condensin I and II complexes act together to compact and organise DNA into rod-like chromosomes during mitosis. This genomic reorganisation affords chromosomes the necessary rigidity and strength to withstand the mechanical forces involved during mitotic segregation, ultimately ensuring genetic material is accurately and faithfully transmitted to each new daughter cell. However, the manner in which condensins function over the course of physiological mammalian development, and how their functions can change and adapt across the various tissues and cell types that arise during this process, is not readily understood and can be difficult to study due to the essential nature of the complexes.

Targeted protein degradation (TPD) is a useful method to directly study protein function whilst minimising the potential complications that commonly arise after deleting or degrading alternative upstream targets such as DNA and RNA. Proteins of interest can be tagged with ‘degron’ peptides using genome editing strategies such as CRISPR-Cas9. These peptides can then be conditionally degraded via the addition of a small molecule ligand. Such conditional methods facilitate the study of essential proteins like the condensin complexes, allowing cells and organisms to grow and develop normally in the presence of the essential protein of interest, before initiating protein depletion. The auxin-inducible degron (AID) system is a commonly used TPD method, and has been extensively and successfully utilised across many model systems, from yeast to human cells. However, AID functionality and efficacy in transgenic mammalian organisms is an area that has remained relatively underexplored.

To increase our collective understanding of how the AID system functions in mammalian models, in addition to generating a transgenic mouse embryonic stem cell (mESC) line, I have used CRISPR-Cas9 to generate transgenic mouse lines in which essential condensin subunits (NCAPH and NCAPH2) have been endogenously tagged with a mini-AID (mAID) peptide and the fluorescent protein Clover, and subsequently combined with the expression of a transgenic F-box protein, OsTIR1, that facilitates the degradation process.

The initial exploration of the AID system in an Smc2-mAID-Clover transgenic mESC line revealed that the mESCs could tolerate the transgenic modification of a condensin subunit, and that indole-3-acetic acid (IAA)-mediated degradation was extremely rapid and efficient in this mammalian system (70- 80% depletion after 1-5 hour IAA treatment). This depletion was sufficient to completely ablate cell growth, likely due to a mitotic block, but no evidence for interphase roles for the complexes were uncovered in the limited number of assays performed using the cell line.

Both Ncaph- and Ncaph2-mAID-Clover homozygous mice were fertile and viable, and the fluorescent Clover tag facilitated the monitoring of NCAPH and NCAPH2 expression in a variety of different tissues, revealing that expression did not seem to fluctuate greatly across actively dividing cells. Introducing the OsTIR1 allele to both lines facilitated the rapid degradation of endogenous AID-tagged condensin subunits upon the addition of IAA. Efficient depletion was observed across multiple cell types, both under ex vivo and in vivo conditions. Building upon preliminary work performed using the Smc2-tagged mESC line, it was discovered that IAA dosage, cell type, and OsTIR1 and AID-tagged protein expression levels all had an effect on the kinetics and efficacy of IAA-induced protein degradation. The advantages of the AID system were leveraged to probe the requirement for NCAPH and NCAPH2 across mammalian development, to reveal that the mitotic requirements for these proteins may vary in a cell-type-specific manner. Finally, I was able to show the IAA-mediated degradation was rapid, efficient and specific when the ligand was introduced to live animals.

Together, this work represents the generation, validation and characterisation of the first germline AID-mouse lines in which functional, endogenously expressed AID-tagged proteins can be efficiently and rapidly degraded upon IAA addition, and an initial exploration of how condensin function can vary during mammalian development. It is hoped that the knowledge gleaned from this body of work can be used as a foundation for future studies to build on, in order to fully understand the intricacies of the AID system, and indeed how the condensin complexes function, across the variety of cellular environments that exist in a mammalian organism.