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CRISPR Knockout cell models are moving science forward

Jun 1, 2017 4:34:06 PM No Comments

The dilemma of when to invest in new technology

Researchers in the life sciences community are constantly walking a fine line in assay development. On one side is the accuracy, specificity and reproducibility borne from use of a well-established tool; i.e., a tool that has been on the market for a long time. Put another way, there is a level of comfort in using the same products for many years - in science as in the rest of life.

On the other side is the importance of finding the most efficient, cost-effective methods to carry out experiments. Doing so often means taking a chance on a new product, running it alongside existing methods to compare. Of course, it’s not just cost-effectiveness that necessitates making changes; simply keeping up can mean bringing in a new product that incorporates new advances. The outcome, hopefully, is better results faster, at lower cost.

And yet, inertia is a challenging force to overcome, and there is always a tendency to maintain the status quo. Particularly, as noted above, when so much rides on maintaining consistent protocols.

Here at Horizon, our scientists have built a remarkable new tool in the HAP1 cell line to facilitate researcher's access to CRISPR technology. These knockout cell lines allow researchers to quickly validate their gene or target of interest, without having to invest time and resource in developing in-house CRISPR technology.

HAP1 knockout cells for antibody validation_SLC30A6.png

HAP1 and HAP1 cells gene-edited to knockout SLC30A6 (HAP1_SLC30A6, catalogue number: HZGHC002784c010) with the HPA antibody HPA057328 targeting SLC30A6 demonstarting the specificity of this antibody. The samples were prepared in parallel using the same antibody dilutions and reagents, and both images are acquired with the exact same settings. Images curtesy of Dr Emma Lundberg, Cell Profiling facility. KTH Royal Institute of Technology.

We believe that, for its designated applications, HAP1 cells are more than worth adding into a lab’s toolbox. However, our opinion only takes things so far. So we set out to ask a few scientists who have published using the HAP1 cells about their work, and how the cells played a role in their investigations.

Bridging the gap when patient derived cells are not sufficient

Dr. Sandra Pohl, of the University Medical Center Hamburg-Eppendorf, has spent much of her career studying targeting of proteins to the lysosome. Of particular interest to her group is N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), a hexameric protein that localizes to the Golgi. This enzyme catalyzes production of the molecular signals required to target lysosomal enzymes to their final destination. Two genes, GNPTAB and GNPTG encode the three subunits (of which there are two each in the full multimeric protien). Mutations in these genes cause the autosomal recessive diseases mucolipidosis type II and/or III, in which lysosomes accumulate material that should be degraded due to the lack of sufficient (or functional) lysosomal enzymes.

While the genetic mutations surrounding mucolipidoses are reasonably well-understood, the structural consequences of those mutations were not. Dr. Pohl’s group is working to remedy that gap. Over the past several years, they have been investigating how the various GlcNAc-1-phosphotransferase subunits (α, β and 𝛾) interact with each other and binding sites required for a functional protein. Additionally, they are intrigued by the enzyme’s ability to “recognize and distinguish lysosomal enzymes from hundreds of other glycoproteins synthesized in the secretory pathway.”

Recently, the group found that the binding of the γ and α subunits to each other “is required for efficient Glc-NAc-1-phosphotransferase activity.” To reach this conclusion (along with others from additional papers), Dr. Pohl’s team utilized the HAP1 cells to fill a significant experimental gap: cells carrying mutated forms of GlcNAc-1-phosphotransferase from human patients “are limited in availability and they grow very slowly.” Furthermore, those cells can only be maintained for a few passages. Therefore, as in many avenues of research, primary cell culture can create frustrating delays in experimental progress.  Dr. Pohl’s group turned to HAP1 cells to bridge the gap due to the lack of viable cell lines with defective forms of GlcNAc-1-phosphotransferase. For her group, the HAP1 cells fit in to an overall experimental approach that included knockout mouse models. The line, she said, “are always a good choice when KO mouse models are not available or if human cells are preferred.”

Jumping roadblocks caused by inflexible biological models

In an entirely different avenue of research, Dr. Pavel Ivanov of Harvard Medical Center and Brigham and Women’s Hospital, investigates stress granule formation in cells. Stress granules are a bit of an odd phenomenon. They are tied closely to translation and gene expression, effectively reprogramming cells to prevent pro-apoptotic signals in order to promote cell survival. Stress granules  sequester mRNA of many housekeeping genes, which significantly ramps down translation and conserves energy (translation can at times take up to 80% of a cell’s energy needs). Dr. Ivanov also notes that the progression of several diseases, including ALS, is marked at the cellular level by problems in stress granule formation or disassembly. Therefore, getting a better understanding of these transient and somewhat elusive structures is quite important.

Notably, while numerous stresses can induce stress granules, researchers have only assumed, rather than demonstrated, that the granules formed under different circumstances are the same. Work from Dr. Ivanov’s lab was the first to explore this issue in detail, as described in a Journal of Cell Science paper published in March, 2017. (They also recently published a new paper with a protocol to categorize cytoplasmic foci in mammalian cells as stress granules.) Additionally, his group has sought to close an existing gap in understanding of the steps that occur between translational arrest and formation of microscopically visible stress granules.

Dr. Ivanov’s group began working with HAP1 cells to add context to recent work in the field that used in vitro experiments to look at protein-protein interactions thought to induce stress granule formation. Because they are biochemical interactions, he said that “most of those studies are done in vitro and their relevance to in vivo situations is under intensive investigation.” Rabbit reticulocyte lysates are also standard in the field; however, their relevance is notably tempered due to enrichment in certain proteins that may skew results and because they cannot be genetically manipulated.

Consistency of CRISPR gene edited models versus siRNA 

For both groups, the advantage of having a turnkey cellular system to investigate the consequences of knockout/mutation of genes of interest was at the heart of selecting HAP1 cells. As true knockouts, generated by CRISPR-Cas9, the line offers more consistency than other common methods for reducing gene expression. Dr. Pohl has in the past used siRNA in HeLa cells to complement the mouse models when available. In addition, strong comparisons can be made between HAP1 cells and those found in mucolipidosis patients. According to Dr. Pohl, HAP1 cells “show the same biochemical behavior as patient cells and are therefore a valid cell culture model of the disease.” This also means that the group can - and plans to - apply the HAP1 line to many varied studies going forward.

Quick access to the advantages of CRISPR gene-editing technology

Of course, CRISPR-Cas9 technology is not available only to corporate labs and commercial suppliers such as Horizon. Many academic labs can and do utilize the system for creating in-house knockouts. And RNAi protocols remain a staple. However, in the case of Dr. Ivanov’s group, it became more efficient and cost effective to purchase existing lines from Horizon. The decision was one of convenience plus technical utility. He said, “generally speaking, CRISPR/CAS9 works well for genetic editing in our standard in-house osteosarcoma U2OS cell line. However, selection of clones with gene knock-out can be problematic.” Additionally, “the main plus for us was that a panel of HAP1 cells with knockout of four eIF2α kinases was immediately available.” (Phosphorylation of eIF2α is a key step in translation initiation.)

Opening up new possibilities for future research

For researchers like Dr. Pohl, the benefits of HAP1 cells are significant enough that it was worth breaking inertia of doing in-house knockdowns and moving to a new tool. Certainly, the addition of this line did not obviate the need for many of the tools already in place, such as mouse models. However, the value of confirmed knockout of GNPTG, along with the efficiency gained through the improved culturability of HAP1 cells relative to primary patient cells, was more than sufficient to attract the group to the relatively new system developed here at Horizon. With results such as the publications from the Pohl group that this tool does indeed fulfill the need for better results faster, and at lower cost.

Dr. Ivanov also noted that HAP1 cells are one of many tools they will use going forward.  His lab is working on the next round of experiments taking advantage of HAP1 cells to look at the molecular underpinnings of stress granule formation and disassembly: “We plan using HAP1 cells for genetic manipulation of non-coding RNAs or untranslated regions of specific mRNAs, namely to introduce mutations in specific regions of such RNAs.”

Further information on HAP1 cells and how they improve research programme outcomes:

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