gene editing

CRISPR/Cas9 Knock-In Tagging: Simplifying the Study of Endogenous Biology

Posted on by Ken Doyle

Understanding the expression, function and dynamics of proteins in their native environment is a fundamental goal that’s common to diverse aspects of molecular and cell biology. To study a protein, it must first be labeled—either directly or indirectly—with a “tag” that allows specific and sensitive detection.

Using a labeled antibody to the protein of interest is a common method to study native proteins. However, antibody-based assays, such as ELISAs and Western blots, are not suitable for use in live cells. These techniques are also limited by throughput and sensitivity. Further, suitable antibodies may not be available for the target protein of interest.

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Targeted Gene Modification in Prairie Voles Using CRISPR and pGEM®-T Easy Vectors

Posted on by Leah Cronan

As the number of children diagnosed with autism spectrum disorder (ASD) continues to rise, the search for a cause continues. Scientists have been studying genetically modified oxytocin receptors, which have shown promise as a target for studying ASD-related behaviors. One of the obstacles to designing robust scientific experiments for investigating potential ASD causes or treatments is the lack of a truly appropriate model organism for social behaviors in humans (1). Sure, there are the traditional lab rats and lab mice that demonstrate a certain level of social behaviors. However, there has been a loss of natural social behaviors in common lab mice strains because of the reduction in genetic complexity from inbreeding and adaptation to captivity (2). These animals cannot fully represent the depth of human social behaviors, including the ability of humans to form lasting social bonds (1).

Enter: The prairie vole (Microtus ochrogaster).

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Discussing the Future of Gene Editing at CRISPRcon Midwest

Posted on by Mariel Mohns
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Walking in to the first session at CRISPRcon Midwest.

Last week, a diverse group of stakeholders attended CRISPRcon Midwest, hosted by the Keystone Policy Center and the University of Wisconsin–Madison. The goal of the day-long conference was to emphasize the importance and value of gene editing technology, and how it must be communicated deliberately between scientists, the public, policymakers, and other stakeholders.

Julie Shapiro, Senior Policy Director of Keystone Policy Center, acted as Emcee for the event. Given the diverse group of attendees, she mentioned in her opening remarks that the event organizers were “seeking conversation, not consensus” and emphasized the “power of respectful dialogue.” A slide overhead showcased the ground rules for the day, which included statements such as “dare to listen, dare to share, and dare to disagree.”

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Word cloud generated from live polling results at CRISPRcon Midwest.

CRISPRcon aimed to included voices beyond those represented by keynote speakers and panelists, so they incorporated live polling through an online app to keep the audience engaged and an active participant in the conversations throughout the day. From the opening remarks, it was clear that this conference would not just deliver on its promise of thoughtful conversation about the science, but build further understanding about the societal impacts of a rapidly advancing technology.

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

Posted on by Sara Klink

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

CRISPR: Gene Editing and Movie Madness

Posted on by Alyssa Bear

There are new developments in genetics coming to light every day, each with the potential to dramatically change life as we know it. The increasingly controversial gene editing system, dubbed CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), is at the root of it all. Harnessed for use in genome editing in 20131, CRISPR has given hope to researchers looking to solve various biological problems. It’s with this technology that researchers anticipate eventually having the means to genetically modify humans and rid society of genetic disorders, such as hemophilia. While this is not yet possible, the building blocks are steadily being developed. Most recently, two groundbreaking studies concerning CRISPR have been released to the public. Continue reading “CRISPR: Gene Editing and Movie Madness”

A Crash Course in CRISPR

Posted on by Darcia Schweitzer

CRISPR is a hot topic right now, and rightly so—it is revolutionizing research that relies on editing genes. But what exactly is CRISPR? How does it work? Why is everyone so interested in using it? Today’s blog is a beginner’s guide on how CRISPR works with an overview of some new applications of this technology for those familiar with CRISPR.

Introduction to CRISPR/Cas9

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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) were discovered in 1987, but it took 30 years before scientists identified their function. CRISPRs are a special kind of repeating DNA sequence that bacteria have as part of their “immune” system against invading nucleic acids from viruses and other bacteria. Over time, the genetic material from these invaders can be incorporated into the bacterial genome as a CRISPR and used to target specific sequences found in foreign genomes.

CRISPRs are part of a system within a bacterium that requires a nuclease (e.g. Cas9), a single guide RNA (sgRNA) and a tracrRNA. The tracrRNA recruits Cas9, while sgRNA binds to Cas9 and guides it to the corresponding DNA sequence of the invading genome. Cas9 then cuts the DNA, creating a double-stranded break that disables its function. Bacteria use a Protospacer Adjacent Motif, or PAM, sequence near the target sequence to distinguish between self and non-self and protect their own DNA.

While this system is an effective method of protection for bacteria, CRISPR/Cas9 has been manipulated in order to perform gene editing in a lab (click here for a video about CRISPR). First, the tracrRNA and sgRNA are combined into a single molecule. Then the sequence of the guide portion of this RNA is changed to match the target sequence. Using this engineered sgRNA along with Cas9 will result in a double-stranded break (DSB) in the target DNA sequence, provided the target sequence is adjacent to a compatible PAM sequence.

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