A Light Switch for Gene Editing

NEWSLETTER

Cell Crunch (Issue 2021.03.08)

A Light Switch for Gene Editing: I’m a sucker for CRISPR, but it has its drawbacks. The Cas9 protein chomps away at DNA until it is removed, or its guide RNA is destroyed. CRISPR-Cas9 can also exert a “burden” on cells (perhaps due to off-target cutting of DNA), causing them to grow more slowly.

There are a lot of ways to shut down Cas9, but none are ideal. Anti-CRISPR proteins can switch off DNA-editing, for example, but they have to be expressed in the cell at a Goldilocks level. Chemicals can also be used, but that demands careful dosage to avoid harming the cells.

Their approach is simple: Within the crRNA portion of a guide RNA — the bit that actually binds to the DNA target — they implanted an internal, photocleavable linker molecule, called 2-nitrobenzyl. The linker molecule is placed five bases away from the end of the crRNA, and if you blast the cells with light (365 nm wavelength), the linker is obliterated and the end of the guide RNA falls off. Cas9 stops cutting.

This photocleavable approach is very fast, with nearly total Cas9 deactivation within one minute of light exposure. The authors write that their approach is “the fastest and most complete strategy for Cas9 deactivation, improving on prior arts by at least an order of magnitude in both speed and residual activity.”

Why It Matters: Methods to switch off Cas9 have historically worked by adding an additional component to cells — anti-CRISPR proteins, chemicals, siRNA sequences, and so forth. This new approach for Cas9 deactivation is non-invasive, requiring only light and a modified guide RNA. It could be used to reduce the toxicity that Cas9 exerts on living cells, or to answer gene-editing questions that have a…

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