Last year was an exciting one for Addgene as we introduced our long-awaited viral service, but we haven’t forgotten about our plasmids! Now, we’re improving our quality control processes using next-generation sequencing (NGS) services provided by seqWell. This new QC process will bring you full sequence data for new plasmids entering the repository. Read on to learn more about how this process works and what you can expect to see on our plasmid pages.
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.
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!
When we talk about CRISPR applications, one negative always comes up: the low editing efficiency of homology-directed repair (HDR). Compared to the random process of non-homologous end joining, HDR occurs at a relatively low frequency, and in nondividing cells, this pathway is further downregulated. Like all CRISPR applications that use wild-type Cas9, editing by HDR also has some potential for off-target cleavage even when gRNAs are well designed. Rather than try to improve HDR, Addgene depositor David Liu’s lab created new Cas9 fusion proteins that act as “single base editors.” These fusions contain dCas9 or Cas9 nickase and the rat cytidine deaminase APOBEC1, which can convert cytosine to uracil without cutting DNA. Uracil is subsequently converted to thymine through DNA replication or repair. Komor et al. estimate that hundreds of genetic diseases could be good targets for base editing therapy, not to mention the potential basic and preclinical research applications. Read on to learn about this new way to make point mutations using CRISPR without double-stranded breaks.
In 2015, Feng Zhang’s lab characterized two Cpf1 nucleases, distant cousins of well-known Cas9. Cpf1 cleaves DNA in a staggered pattern and requires only one RNA rather than the two (tracrRNA and crRNA) needed by Cas9 for cleavage. Now, two new studies show that Cpf1 displays lower off-target editing than Cas9, confirming that this protein is well suited for genome editing.