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Originally published Feb 14, 2017 and updated Jun 24, 2020.

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.

Promoters control the binding of RNA polymerase and transcription factors. Since the promoter region drives transcription of a target gene, it therefore determines the timing of gene expression and largely defines the amount of recombinant protein that will be produced. Many common promoters like T7, CMV, EF1A, and SV40, are always active and thus referred to as constitutive promoters. Others are only active under specific circumstances. In a previous post, we discussed inducible promoters, which can be switched from an OFF to an ON state, and how you might use these in your research. Today, we’ll look at repressible promoters, which can be switched from an ON to an OFF state, as well as repressible binary systems commonly used in Drosophila.

RNA-editing Cas13 enzymes have taken the CRISPR world by storm. Like RNA interference, these enzymes can knock down RNA without altering the genome, but Cas13s have higher on-target specificity. New work from Konermann et al. and Yan et al. describes new Cas13d enzymes that average only 2.8 kb in size and are easy to package in low-capacity vectors! These small, but mighty type VI-D enzymes are the latest tools in the transcriptome engineering toolbox.

CRISPR genome editing has made it easier to create knockout alleles in a variety of species, including the standard laboratory mouse. It’s also made targeted insertions relatively simple in C. elegans and bacteria. But CRISPRing typical mouse models, including creating Cre-dependent conditional alleles, has remained a challenge. Enter Easi-CRISPR: a method that harnesses the power of ssDNA donor molecules for homology directed repair. Using long ssDNA donors, the Gurumurthy and Ohtsuka groups have obtained an average knock-in efficiency of 30-60%. This is much more favorable than previous methods yielding 1-10% knock-in. Read on to learn how you can make CRISPR mouse model generation easi-er!

In order to bind DNA, Cas9 and other CRISPR enzymes require a short PAM sequence adjacent to the targeted sequence at the locus of interest. SpCas9’s 3’ NGG PAM occurs frequently in GC-rich genomes, but a PAM is not always available near the locus you’d like to modify. To tackle the PAM problem, researchers have engineered alternative Cas9s binding distinct PAM sequences. Now, Hu et al., working in David Liu’s lab, have gone one step further, using directed evolution to create xCas9, an enzyme recognizing a broad range of PAMs like NG, GAA, and GAT, but also displaying increased editing specificity. We’re excited to learn more about xCas9 - here’s what we know so far!