Targeted protein degradation phenotypic studies using HaloTag CRISPR/Cas9 endogenous target tagging and HaloPROTAC

Biologists currently have several options in their arsenal when it comes to gene silencing. if you want to completely vanquish the gene in question, you can use CRISPR to knock the gene out completely. This is a great way to completely eliminate the gene, and hence compare cell phenotypes with and without the gene, but it’s less good if the gene is essential and the cells won’t grow without it in the first place. 

Otherwise you can use RNA interference, where small pieces of RNA that complement the mRNA for that gene are introduced to the cell, with the overall effect of blocking transcription of that gene’s mRNA, hence silencing it. However, this method suffers from side effects and varying levels of gene knockdown efficiency. Moreover, it does not vanquish existing protein, it just stops more from being produced.

For some protein targets, PROTACS (proteolysis targeting chimera) are available. These are small molecules (admittedly often quite large small molecules) that tether your protein of interest to an E3 ligase, hence causing it to get degraded by the cell’s proteosomal machinery. However PROTACs are only an option for the few targets for which they have been developed, and developing a new PROTAC for every gene you wish to knockdown is… well, good luck if you do decided to go down that route.

This paper (https://pubmed.ncbi.nlm.nih.gov/33332748/) introduces another option: CRISPR mediated degron fusion to an endogenous protein of interest, and subsequent dose-dependent degradation by addition of a small molecule. The degron here is a 297 amino acid soluble globular protein of bacterial origin called HaloTag, which can form an irreversible covalent bond with a chloroalkane-containing compound; the small molecule is question is HaloPROTAC: a PROTAC with a VHL (an E3 ligase) binder at one end and a chloroalkane at the other. So, much like regular PROTACs, this molecule induces the degradation of your protein of interest by tethering it to an E3 ligase and getting it ubiquitinated then destroyed. But the key difference here is that you can do this for any protein to which you have CRISPR-ed the HaloTag degron.

A few things to take into account:

  • This PROTAC does not work catalytically like other, non-covalent PROTACs (but is still an affective degrader)
  • on the flip side, this PROTAC also does not exhibit the Hook effect, as seen for non-covalent PROTACs
  • The protein you choose as your target will still be expressed endogenously, but with a 297 amino acid domain fused onto it. Depending on your choice of target, this may have serious implications for its regular function and hence the ‘wild type’ phenotype (i.e. no added HaloPROTAC)

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