Have you been overwhelmed by the number of CRISPR articles published in 2017? PubMed alone has cited over 3,000 CRISPR publications in 2017! We wanted to save you from having to sieve through the databases by asking our experts to select what they thought were the most important CRISPR publications from last year.
For the first time, Human Knockout cell lines are readily available for scalable reverse genetic screening.
We speak to Horizon's Head of Innovation, Dr Tilmann Bürckstümmer about the application of reverse genetic screening using a combination of new technologies.
Gapp et al. (2016) in Molecular Systems Biology
Revealing the role of E3 ubiquitin ligases in DNA damage repair
One of the diverse new uses for the HAP1 cell line, one that has begun to draw significant attention, is in the field of DNA damage repair. A recent paper from Minoru Takata’s group highlights this important application of this relatively new tool.
What more do we know since last year?
As more and more papers are published using models generated by CRISPR/Cas9 editing, and new and exciting applications for the CRISPR/Cas system continue to be invented, the potential for off-target editing continues to be discussed. We published an article on our blog a year ago which explains the potential for off-target editing with CRISPR/Cas9 and summarised some of the literature on this topic, and thought this was a good time for an update.
...are defined as genes that are critical for the survival of an organism. These are considered to be genes that are absolutely required for the cell to grown, proliferate and survive. Deletion of an essential gene from a cell eventually leads to the death of this cell or a severe proliferation defect. As a consequence, it is impossible to generate cells with a knock-out or deletion of essential genes.
In a breakthrough study, Blomen et al. (Science, 2015) used extensive mutagenesis to describe the complete set of essential genes in the human haploid cell line Hap1.
Mutations were generated by the random introduction of a gene-trap cassette that interferes with correct splicing.
A revolution is under way in functional genomics which is spearheaded by the CRISPR-Cas9 system and its application to pooled genetic screening. Remarkable new tools, made possible by dCas9, are coming to fruition that will allow for a new kind of interrogation of gene function, allowing us to ask more sophisticated questions about the biology of drug targets.
January 2013 was marked by a major breakthrough in genome engineering. Four labs simultaneously engineered the bacterial and archaeal CRISPR-Cas9 system to induce precise cleavage at mammalian genomic loci1–4. Within a year’s time, two back-to-back papers documented the application of CRISPR-Cas9 knockout technology to forward genetic screening5,6. These studies showed not just proof of concept of a new technology, but a spectacular jump in what is possible within functional genomics. Many studies have since capitalised on these discoveries and several publications, including from Horizon7, have demonstrated screening platforms with even greater precision and performance.
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 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.
Research conducted by Jian-Hua Luo, M.D., Ph.D. of the Pittsburgh School of Medicine is the first time gene editing has been used to specifically target cancer fusion genes, which are hybrid genes discovered in a wide array of solid tumors1.
These hybrid genes, formed from two previously separate genes, produce abnormal proteins that can be a catalyst to faster and/or further cancer growth. In patients this causes far more invasive cancers, reducing life expectancy and survival chances.
Read our blog on how new methods increase precision in protein visualization
Here we describe some of the great solutions that are coming out of recent advances using CRISPR CAS technology that give more precise and physiological results for protein visualization. In our previous blog (see link at end of article), we discussed the some of the difficulties with traditional methods for protein tracking and localization. One of the main causes of variability and wasted resources is the lack of standards for antibody quality.
To be useful, an antibody must:
Have a high signal to noise ratio
Be validated for the assay at hand
Efforts to reduced non-specific antibodies in both industry and academia
From an industry standpoint, numerous organizations and commercial suppliers have created (or are creating) programs to ensure that the above criteria are met for each new antibody brought to market.