All You Need is a Tether: Improving Repair Efficiency for CRISPR-Cas9 Gene Editing

Ribonucleoprotein complex with Cas9, guide RNA and donor ssDNA. Copyright Promega Corporation.

With the advent of genome editing using CRISPR-Cas9, researchers have been excited by the possibilities of precisely placed edits in cellular DNA. Any double-stranded break in DNA like that induced by CRISPR-Cas9 is repaired by one of two pathways: Non-homologous end joining (NHEJ) or homology-directed repair (HDR). Using the NHEJ pathway results in short insertions or deletions (indels) at the break site, so the HDR pathway is preferred. However, the low efficiency of HDR recombination to insert exogenous sequences into the genome hampers its use. There have been many attempts at boosting HDR frequency, but the methods compromise cell growth and behave differently when used with various cell types and gene targets. The strategy employed by the authors of an article in Communications Biology tethered the DNA donor template to Cas9 complexed with the ribonucleoprotein and guide RNA, increasing the local concentration of the donor template at the break site and enhancing homology-directed repair. Continue reading

Biotechnology From the Mouths of Babes

As a science writer, much of my day entails reviewing and revising marketing materials and technical literature about complex life science research products. I take for granted the understanding that I, my colleagues and our customers have of how these technologies work. This fact really struck me as I read an article about research to improve provider-patient communication in healthcare settings.

The researchers completed an analysis revealing that patient information materials had an average readability at a high school level, while the average patient reads at a fourth-grade level. These findings inspired the researchers to conduct a study in which they enlisted the help of elementary students to revise the content of the patient literature after giving them a short lesson on the material.

The resulting content did not provide more effective ways to communicate indications, pre- and post-op care, risks or procedures—that wasn’t really the point. Instead, the study underscores the important connection between patient literacy and health outcomes. More specifically, a lack of health literacy is correlated with poor outcomes and increased healthcare costs, prompting action from the US Department of Health & Human Services.

While healthcare information can be complex and full of specific medical terminology, I recognized that a lot of the technical and marketing information we create for our products at Promega has similar features. Wouldn’t it be interesting to find out how descriptions of some of our biggest technologies translate through the eyes and mouths of children?

After enlisting some help from my colleagues, I was able to catch a glimpse of how our complex technologies are understood by the little people in our lives. The parents and I explained a technology and then had our child provide a description or drawing of what they understood. Continue reading

Shining Stars: Cool NanoLuc® Plasmid Constructs Available Through the Addgene Repository

Researchers having been sharing plasmids ever since there were plasmids to share. Back when I was in the lab, if you read a paper and saw an interesting construct you wished to use, you could either make it yourself or you could “clone by phone”.  One of my professors was excellent at phone cloning with labs around the world and had specific strategies and tactics for getting the plasmids he wanted. Addgene makes this so much easier to share your constructs from lab to lab. Promega supports the Addgene mission statement: Accelerate research and discovery by improving access to useful research materials and information.  Many of our technology platforms like HaloTag® Fusion Protein, codon-optimized Firefly luciferase genes (e.g., luc2), and NanoLuc® Luciferase are present in the repository. We encourage people to go to Addgene to get new innovative tools. Afterall, isn’t science better when we share?

I’d like to focus on some tools in the Addgene collection based on NanoLuc® Luciferase (NLuc).  The creation of NanoLuc® Luciferase and the optimal substrate furimazine is a good story (1).  From a deep sea shrimp to a compact powerhouse of bioluminescence, NLuc is 100-fold brighter than our more common luciferases like firefly (FLuc) and Renilla (RLuc) luciferase.  This is important not so much for how bright you can make a reaction but for how sensitive you can make a reaction.  NLuc requires 100-fold less protein to produce the same amount of light from a Fluc or RLuc reaction.  NLuc lets you work at physiological concentrations.  NLuc is bright enough to detect endogenous tagged genes generated through the CRISPR/Cas9 knock-in.  NLuc is very inviting for endogenous tagging as it is only 19kDa.  An example is the CRISPaint-NLuc construct (Plasmid #67178) for use in the system outlined in Schmid-Burgk, J.L. et al (2).

Two applications of NanoLuc® Technology have caught my attention through coupling the luciferase with fluorescent proteins to make better imaging reporters and biosensors. Continue reading

Probing RGS:Gα Protein Interactions with NanoBiT Assays

gpcr_in_membrane_on_white2When I was a post-doc at UT Southwestern, I was fortunate to interact with two Nobel prize winners, Johann Deisenhofer and Fred Gilman.  Johann once helped me move a -80°C freezer into his lab when we lost power in my building. I once replaced my boss at small faculty mixer with a guest speaker and had a drink with Fred Gilman and several other faculty members from around the university. Among the faculty, one professor had a cell phone on his belt, an odd sight in 1995. Fred Gilman asked him what it was and why he had it. It was so his lab could notify him of good results anytime of the day. Fred laughed and told him to get rid of it – if it’s good data, it will survive until morning.

I was reminded of this story when I read a recent paper by Bodle, C.R. et al (1) about the development of a NanoBiT® Complementation Assay (2) to measure interactions of Regulators of G Protein Signaling (RGS) with Gα proteins in cells. (Fred Gilman was the first to isolate a G protein and that led to him being a co-recipient of the Nobel Prize in 1994). The authors created over a dozen NanoBiT Gα:RGS domain pairs and found they could classify different RGS proteins by the speed of the interaction in a cellular context. The interactions were readily reversible with known inhibitors and were suitable for high-throughput screening due to Z’ factors exceeding 0.5. The study paves the way for future work to identify broad spectrum RGS domain:Gα inhibitors and even RGS domain-specific inhibitors. This is the second paper applying NanoBiT Technology to GPCR studies (3).

A Little Background…
A primary function of GPCRs is transmission of extracellular signals across the plasma membrane via coupling with intracellular heterotrimeric G proteins. Upon receptor stimulation, the Gα subunit dissociates from the βγ subunit, initiating the cascade of downstream second messenger pathways that alter transcription (4). The Gα subunits are actually slow GTPases that propagate signals when GTP is bound but shutdown and reassociate with the βγ subunit when GTP is cleaved to GDP. This activation process is known as the GTPase cycle. G proteins are extremely slow GTPases. Continue reading

Optimizing Antibody Internalization Assays: pHAb Dyes

22255190_pHabAmine_3D_image_050615-final-largePromega has recently developed a method that allows antibodies to be screened for their internalization properties in a simple, plate-based format. The method uses pH sensor dyes (pHAb dyes), which are not fluorescent at neutral pH but become highly fluorescent at acidic pH. When an antibody conjugated with pHAb dye binds to its antigen on the cancer cell membrane, the antibody-dye-antigen complex is not fluorescent, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops, and the dye becomes fluorescent.

To demonstrate the broad utility of the pHAb dye for receptor mediated antibody internalization, two therapeutic antibodies, trastuzumab and cetuximab,which bind to HER2 and EGFR respectively, were selected for a case study (1). Both the antibodies, which are known to internalize were labeled with pHAb dyes using amine or thiol chemistry.

Parameters such as the impact of dye–to-antibody ratio on the antigen–antibody binding, change in fluorescence as a function of pH of free dye and labeled dye, and labeled antibody internalization as a function of pHAb conjugated antibody concentration were evaluated.

The results indicate that pHAb dyes are pH sensitive fluorescent dyes that enable the study of receptor-mediated antibody internalization.Internalization assays can be performed in a plate-based homogeneous format and allow endpoint assays as well as real-time monitoring of internalization. They further show that internalization can be monitored even at a very low amount of antibody which is very important during the early monoclonal antibody development phase when the amount of sample is limited and the antibody concentration in the samples is low. a complimentary approach, they  also showed that a secondary antibody labeled with pHAb dye can be used instead of labeling primary antibodies.

Literature cited

Nath, N. et al. (2016) Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye J.  Immunol. Methods epub ahead of print

Studying Mitochondrial Fission with NanoBiT Complementation Assay

Vote for NanoBiT™ in the Scientist's Choice Awards and get a chance to win a $500.00 US Amazon voucher from Select Science.

Vote for NanoBiT™ in the Scientist’s Choice Awards and get a chance to win a $500.00 US Amazon voucher from Select Science.

It’s a new year. Whether you’re a self-improvement fanatic or just ready for good things to start happening, you’ve got a plan. You might be changing up an old exercise routine or trying a new cooking technique.

And at work, you are digging deeper; this is the year you illuminate the protein interactions that you’ve previously not been able to visualize.

Good news. There is a new protein complementation assay that can help. Studying-mitochondrial-fission-poster

About NanoBiT
NanoBiT™ Complementation Reporter is a recently developed protein interaction assay that features the improved NanoLuc® luciferase. NanoLuc, originally isolated from a deep sea shrimp, is a small luciferase that provides a much brighter signal than firefly luciferase.

About Split Luciferase Systems
If you’re interrogating two proteins to understand the conditions under which they interact, a split luciferase system enables you to tag each protein with a luciferase subunit. Interaction of the tagged proteins facilitates the complementation of the subunits, resulting in a luminescent signal. Continue reading

Key Advances in PPI Research

small linkedin thumbnailOur understanding of the microscopic world has been shaped by the tools available to monitor and visualize cellular interactions. We “stand on the shoulders of giants” to propel our research to even greater heights. Studying protein-protein interactions (PPI) has proved fruitful for our understanding of cellular metabolism, signal transduction, and more. Scientists are starting to build whole organism interactomes (kindred to the metabolome and genome) that could have huge implications towards understanding and treating disease. Let us take a trip down memory lane to see where we have come from.  Continue reading

NanoBiT™ Assay: Transformational Technology for Studying Protein Interactions Named a Top 10 Innovation of 2015

For three out of the last four years, we have been honored to have one of our key technologies named a Top 10 Innovation by The Scientist. This year the innovative NanoBiT™ Assay (NanoLuc® Binary Technology) received the recognition. NanoBiT™ is a structural complementation reporter based on NanoLuc® Luciferase, a small, bright luciferase derived from the deep sea shrimp Oplophorus gracilirostris.

Using plasmids that encode the NanoBiT complementation reporter, you can make fusion proteins to “report” on protein interactions that you are studying. One of the target proteins is fused to the 18kDa subunit; the other to the 11 amino acid subunit. The NanoBiT™ subunits are stable, exhibiting low self-affinity, but produce an ultra-bright signal upon association. So, if your target proteins interact, the two subunits are brought close enough to each other to associate and produce a luminescent signal. The strong signal and low background associated with a luminescent system, and the small size of the complementation reporter, all help the NanoBiT™ assay overcome the limitations associated with traditional methods for studying protein interactions.

The small size reduces the chances of steric interference with protein interactions. The ultra bright signal, means that even interactions among proteins present in very low amounts can be detected and quantified–without over-expressing large quantities of non-native fusion proteins and potentially disrupting the normal cellular environment. And the NanoBiT™ assay can be performed in real time, in live cells.

The NanoBiT™ assay is already being deployed in laboratories to help advance understanding of fundamental cell biology. You can see how one researcher is already taking full advantage of this innovative technology in the video embedded below:

Visit the Promega web site to see more examples more examples how the NanoBiT™ assay can break through the traditional limitations for studying protein interactions in cells.

You can read the Top 10 article in The Scientist here.

HaloTag® Research Application: Detection of Cancer Biomarkers

10242TAAntibodies labelled with radioisotopes or the sequential administrationof an antibody and a radioactive secondary agent facilitate the in vivo detection and/or characterisation of cancers by positron emission tomography (PET) or by single-photon emission computed tomography (SPECT) imaging.

There are drawbacks to both methods, including prolonged exposure to radiation and  ensuring that both the antibody and the radiolabelled secondary agent are suitably designed so that they bind rapidly upon contact at the tumor.

A recent publication (1) investigated a alternative method utilizing the HaloTag® dehalogenase enzyme HaloTag® is a dehalogenase enzyme (33 kDa) that contains an engineered cavity designed to accommodate the reactive chloroalkane group of a HaloTag® ligand (HTL). Upon entering the enzyme cavity, the terminal chlorine atom rapidly undergoes nucleophilic displacement, and a covalent adduct is formed, effectively anchoring the HaloTag® ligand in a precise location.

Three new HaloTag® ligands were synthesized and each labelled with the SPECT radionuclide indium-111  111In-HTL-1  and the dual-modality HaloTag® ligands,111In-HTL-2 and111;In-HTL-3 containing TMR which allows complementary imaging data).

For the validation of the pretargeting strategy based on these HaloTag® ligands, the target human epidermal growth factor receptor 2 (HER2)was selected. Trastuzumab (Herceptin®) was selected as the primary targeting agent and was modified with HaloTag® protein via the trans-cyclooctene/tetrazine ligation.

All three 111In-labelled HaloTa®g ligands exhibited significantly higher binding to the HER2 expressing when compared to negative controls.

Literature Cited

Knight, J. C et al.(2015) Development of an enzymatic pretargeting strategy for dual-modality imagingChem. Commun. 51, 4055–8.