Plasmids for Endogenous Gene Tagging in Human Cells

Posted by Guest Blogger on Apr 6, 2017 9:02:59 AM

This post was contributed by the gene editing team at the Allen Institute for Cell Science. Learn more by visiting the Allen Cell Explorer at allencell.org and the Allen Institute website at alleninstitute.org.

A classic challenge in cell biology is making sure that what we observe through the microscope represents reality as accurately as possible. This is especially true in the case of protein tagging to elucidate cellular structures. Overexpression methods flood the cell with protein, which can both interfere with a cell’s normal function and result in a ubiquitous background signal that makes it hard to visualize the precise location of the protein or structure of interest.

Endogenous gene tagging is an ideal solution because it allows for tagging and visualization of specific, individual proteins under endogenous regulatory control. But even with the advent of CRISPR/Cas9 technology, inserting large tags into a precise location in the genome is still inefficient, particularly in human cell lines. Furthermore, the quality control necessary to ensure the edited cells are behaving normally can be prohibitively expensive for many labs.

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Topics: Plasmid How To, CRISPR, Techniques

Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

Posted by Leila Haery on Mar 7, 2017 10:30:00 AM

We all know that in the lab there are often little tricks that are essential for experiments but that nobody talks about. After months of troubleshooting, those people who did not tell you that essential thing ask incredulously, “You seriously didn’t add 3 microliters of 5 mM star anise?” This is something I was expecting when I set out to make my first CRISPR/Cas9 gene edit. I wanted to inactivate the gene BRAF (a kinase implicated in several human cancers) in A549 cells (a human lung cancer cell line), armed only with viruses obtained through Addgene’s viral service and the methods sections of scientific articles (gasp). To my delight, not only was I able to make the edits without any reagent-grade endangered Martian chicory root, but considering this is a needle in a haystack type of objective, it was surprisingly easy. It’s true, I CRISPRed. In this post, I’ll summarize the basic steps and analyses, and give what I think are the main tips for each step of performing and analyzing a gene edit using Addgene’s lentiviral CRISPR tools.

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Topics: CRISPR

CRISPRainbow and Genome Visualization

Posted by Mary Gearing on Feb 28, 2017 10:30:00 AM

Colorful CRISPR technologies are helping researchers visualize the genome and its organization within the nucleus, also called the 4D nucleome. Visualizing specific loci has historically been difficult, as techniques like fluorescent in situ hybridization (FISH) and chromosome capture suffer from low resolution and can’t be used in vivo. Some researchers have used fluorescently tagged DNA-binding proteins to label certain loci, but this approach is not scalable for every locus...unlike CRISPR. Early CRISPR labeling techniques allowed researchers to visualize nearly any single genomic locus, and recent advances have allowed scientists to track multiple genomic loci over time using all the colors of the CRISPRainbow.

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Topics: CRISPR, Fluorescent Proteins

CRISPR 101: Editing the Epigenome

Posted by Mary Gearing on Feb 14, 2017 10:44:08 AM

Epigenetic modifications are an additional layer of control over gene expression that go beyond genomic sequence. Dysregulation of the epigenome (the sum of epigenetic modifications across the genome) has been implicated in disease states, and targeting the epigenome may make certain processes, like cellular reprogramming of iPSCs, more efficient. In general, epigenetic chromatin modifications are correlated with alterations in gene expression, but causality and mechanisms remain unclear. Today, targeted epigenetic modification at specific genomic loci is possible using CRISPR, and Addgene has a number of tools for this purpose!

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Topics: CRISPR, CRISPR 101

Truncated gRNAs for Regulating Gene Expression

Posted by Guest Blogger on Jan 10, 2017 10:37:46 AM

This post was contributed by guest bloggers Alissa Lance-Byrne and Alex Chavez, researchers at the Wyss Institute for Biologically Inspired Engineering.

CRISPR/Cas9 technology has revolutionized the fields of molecular biology and bioengineering, as it has facilitated the development of a simple and scalable means of making targeted genetic edits. Cas9 is a DNA binding protein that can be directed to virtually any genetic locus when complexed with an appropriately designed small RNA, or guide RNA (gRNA). The gRNA conventionally contains a 20-nucleotide sequence that is complementary to the target site, or protospacer, in the genome. Native Cas9 has two catalytic domains, each of which cleaves one strand of DNA upon binding the protospacer. The resulting double strand break (DSB) stimulates DNA repair mechanisms that can be exploited to either inactivate a gene or introduce a desired genetic alteration.

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Topics: CRISPR

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