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Originally published Nov 12, 2015 and last updated Aug 20, 2020.

Cas9 can be used to modify any desired genomic target provided that (1) the sequence is unique compared to the rest of the genome and (2) the sequence is located just upstream of a Protospacer Adjacent Motif (PAM sequence). The 3-5 nucleotide PAM sequence serves as a binding signal for Cas9 and this sequence is a strict requirement for Cas9-mediated DNA cleavage.

Originally published Aug 16, 2016 and last updated Aug 6, 2020.

When we talk about CRISPR applications, one negative often comes up: the low editing efficiency of homology-directed repair (HDR). Compared to non-homologous end joining, HDR occurs at a relatively low frequency, and in nondividing cells, this pathway is further downregulated. Rather than try to improve HDR, scientists have developed two classes of base editors: cytosine base editors (CBEs) and adenine base editors (ABEs). 

(There are also RNA base editors, but we’ll just be covering DNA base editors here. To learn more about RNA base editors head over to this blog post: CRISPR 101: RNA Editing with Cas13)

Originally published Nov 30, 2017 and updated Jul 31, 2020.

Cas13 enzymes are quickly becoming major players in the CRISPR field. Just a year after Feng Zhang’s lab identified Cas13a (C2c2) (Abudayyeh et al., 2016) as a RNA-targeting CRISPR enzyme, they adapted Cas13b for precise RNA editing (Cox et al., 2017). This new system, termed REPAIR (RNA editing for programmable A to I (G) replacement) is the first CRISPR tool for RNA editing. Two years after that, the lab published a paper on an RNA editor that allows C to U edits (RESCUE). We’ll walk through how these tools were developed and potential ways you can use it in your research.

Originally published May 23, 2017 and updated Jul 23, 2020.

CRISPR-Cas technology is constantly evolving. Variants of Cas proteins can be used for genome editing, activating gene expression, repressing gene expression, and much more. But there’s one thing that was missing: a way to shut off Cas’s activity. The concern is that the longer Cas remains active in a cell, the greater chances there are for off-target edits to occur. Although methods to switch on Cas activity using light or drugs have been developed, the field lacked an “off-switch” for Cas proteins.

Base editors create specific point mutations in the genome, but they’re inefficient compared to CRISPR/Cas9 edits that rely on double strand DNA breaks. Due to this inefficiency it is crucial for scientists to not only easily identify base editing events in real-time but also enrich for base-edited cells in their experiments. In the past few years, scientists have created an array of base editing reporters that can help you do just that.