28 February 2019

Novel research from the Nilsson lab raises concerns on CRISPR technology for analyzing essential genes

Research from the Nilsson lab at Novo Nordisk Foundation Center for Protein Research (CPR) raises a warning flag for the novel and emerging CRISPR technology when it comes to analyzing essential genes but also provides a novel solution.

The researchers were studying the essential mitotic checkpoint protein Bub1 that plays an important role in controlling the fidelity of chromosome segregation during cell division. To analyze the function of Bub1 in more detail, Assistant Professor Gang Zhang used CRISPR technology to generate Bub1 knock out cell lines by targeting one of the first exons, a common strategy for making knock out cell lines. At first glance, these cell lines appeared to lack Bub1 completely yet were relatively unaffected despite Bub1 being an essential gene.

“I was surprised by this result and also by a number of recent high impact publications from other labs reporting that Bub1 knock out cell lines behaved normally so I decided to analyze all these cell lines more carefully” says Gang Zhang. Strikingly, when Bub1 knock out cell lines were treated with Bub1 RNAi there was a very strong and penetrant phenotype suggesting that the cell lines still expressed residual levels of Bub1 sufficient to support checkpoint signaling (see Figure 1).

Figure 1. Left: The ability of a Bub1 knockout cell line (C) to maintain a mitotic arrest compared to the same cell line treated with a Bub1 RNAi oligo (CR). Right: Bub1 protein levels in the different conditions determined by mass spectrometry. Only in the condition where Bub1 knockout cell lines are treated with Bub1 RNAi is Bub1 completely removed.

“At this point we teamed up with Matthias Mann’s lab at CPR in order to test this hypothesis using mass spectrometry based proteomics, which is very sensitive” says Professor Jakob Nilsson. Indeed, the mass spectrometry analysis showed that all Bub1 knock out cell lines contained residual amounts of Bub1 that could not be detected by western blot or immunofluorescence. Apparently, almost undetectable levels of protein that are likely produced through alternative splicing or use of an alternate start codon can support the function of Bub1. “We were surprised to see how little Bub1 is needed to support its function in the cell. Importantly, this is unlikely to apply only to this protein” says Jakob Nilsson and adds “The conclusion from our study is that researchers have to be very careful with relying on antibody based detection methods for arguing that a null cell line has been generated”.

However, the research conducted by Gang Zhang also provides a simple and powerful new method for studying essential genes. As the CRISPR-generated “knock-out” cell lines expressed very low levels of Bub1 they were extremely sensitive to Bub1 RNAi and this resulted in very penetrant null phenotypes that could be fully complemented with RNAi resistant Bub1 constructs. The researchers then successfully used this new approach to study the function of another essential protein in the pathway, Rod, revealing that this method could be extended to other essential genes and give novel insight.

The penetrant removal of Bub1 and Rod allowed the researches to settle conflicting findings in the field and reveal that the integrated activities of Bub1 and Rod were required for efficient signaling. “Low penetrance of RNAi phenotypes is a common problem that leads to wrongful conclusions but by combining CRISPR technology and RNAi we get very penetrant null phenotypes that can be fully rescued providing a new way of studying proteins” says Jakob Nilsson. The researchers are now using this approach to analyze the function of essential genes in other pathways.

The paper is available from Embo Journal's web site: Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex

Professor Jakob Nilsson