Transcribed RNA can be used to study RNA structure and how it relates to function or how proteins and RNA interact. It can also be used for gene silencing using RNAi (studied more often as a possible therapeutic option) or simply serve as a molecular standard in Real-time RT-PCR. Transcribed RNA is also used in Class 2 Clustered Regularly Interspaced Short Palindromic Repeat systems, or CRISPR.
The CRISPR system, which is naturally occurring in bacteria, has been manipulated to perform gene editing in a laboratory environment. To perform CRISPR in the laboratory environment, you need two main reagents:
The Brains: Guide RNA (gRNA or sgRNA) – Small piece of RNA containing a nucleotide sequence that is capable of binding the chosen Cas Protein, and contains a portion of the sequence that can bind the DNA the researcher intends to modify – the target DNA.
The Brawn: CRISPR-associated endonuclease (Cas Protein) – The protein that cleaves the target DNA; the most popular Cas protein is called Cas9. The Cas protein is guided by the (gRNA).
Synthetic biology has been in the news a lot lately—or maybe it only seems like it because I’m spending a lot of my time thinking about our partnership with the iGEM Foundation, which is dedicated to the advancement of synthetic biology. As the 2019 iGEM teams are forming, figuring out what their projects will be and how to fund them, it seemed fitting to share some of these stories.
Malaria is a life-threatening blood disease that plagues nearly two-thirds of the world’s population. The disease in manifested by parasites of the Plasmodium genus and transmitted to humans through the bite of female Anopheles mosquitoes, which serve as the primary disease vectors. Roughly 200 million people per year are infected with malaria, and approximately 400,000 deaths are reported annually, with children under the age of five comprising the majority of victims.
Africa disproportionately bears the global brunt of this devastating illness, with approximately 92% of all reported cases, as well as 93% of all reported deaths, originating from the continent. This can be partially attributed to the fact that the conditions for transmission are essentially ideal there: the principal vector species Anopheles gambiae are abundant in this region, and not only do they prefer to source their blood from humans over animals, but the mosquitoes also tend to have a longer lifespan, which allows the most common and deadly malaria parasite, Plasmodium falciparum, to complete its life cycle, which contributes to higher disease transmission efficacy.
Though malaria is a preventable disease, often the areas affected most lack access or resources, or are politically unstable, all factors that can contribute to the absence of consistent, functional malaria control programs. Though malaria is also a curable disease, it has long been debated whether eradication was even within the realm of possibility. There are four species of Plasmodium parasites responsible for the pathogenesis of malaria and each exhibit different forms of drug resistance and each responds differently to different medications. This alone makes the prospect of developing a single overarching vaccine for all strains of malaria an improbable achievement and the idea of eradication practically impossible.
Redundancy equips us to survive. We have more than one lung or one kidney for a reason—if one organ in a pair gets damaged, we can still manage if the other is functional. At the cellular level, we have two copies of each chromosome in every non-germline cell. Each copy was inherited originally from a single sperm and ovum, which are “haploid” cells. Consequently, there are two copies of any given gene in non-germline “diploid” cells. In many cases, should one copy of a gene be damaged, the cell can still survive with the other, functional copy of a gene. In plants, this redundancy is common, and many plants exhibit polyploidy. In an extreme example of polyploidy, the large (by bacterial standards) but otherwise unassuming species Epulopiscium contains tens of thousands of copies of its genome.
We have published 130 blogs here at Promega this year (not including this one). I diligently reviewed every single one and compiled a list of the best 8.5%, then asked my coworkers to vote on the top 5 out of that subset. Here are their picks:
Scientific inquiry is a process that is revered as much as it is misunderstood. As I listed to a TED talk about the subject, I was reminded that for the general public the foundation of science is the scientific method—the linear process of making an observation, asking a question, forming an hypothesis, making a prediction and testing the hypothesis.
While this process is integral to doing science, what gives scientific findings credibility and value is consensus from the scientific community. Building consensus is the time-consuming process that includes peer review, publication and replication of results. It is also the part of scientific inquiry that so often leads the public to misunderstand and mistrust scientific findings.
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 “All You Need is a Tether: Improving Repair Efficiency for CRISPR-Cas9 Gene Editing”
February 11 is the International Day of Women and Girls in Science, a reminder that there is still a gender gap in science. Despite the obstacles that women need to overcome, their contributions to field of science have benefited not only their fellow researchers but also their fellow humans. From treatments for diseases to new discoveries that opened up entire fields, women have advanced knowledge across the spectrum of science. Below is a sampling of the achievements of just a few women. What other living female scientist or inventor might you add?
Hate malaria? You can thank Tu Youyou for discovering artemisinin and dihydroartemisinin, compounds that are used to treat the tropical disease and save numerous lives. Her discovery was so significant, she received the 2015 Nobel Prize in Physiology or Medicine. Continue reading “Celebrating Women in Science”
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).
Today’s author extends thanks to Heather Gerard, Intellectual Property Manager, Promega Corporation for contributing her expertise to this post.
Students most often come to the BTC Institute with the primary goal of learning about molecular biology technologies. Our mission is to help them update their experimental tool-box, facilitating more capable studies of DNA, RNA and proteins back in their home laboratories.