Johns Hopkins Researchers Identify CRISPR Dimmer

A naturally occurring system for tuning CRISPR-Cas9 expressing in bacteria, identified in a study published in Cell, could have implications for gene editing therapies as well.

A range of companies are exploring potential gene editing therapies based on a CRISPR-Cas9 system, and the ability to tune the CRISPR cut could prevent off-target editing or control the degree of a target protein’s production.

In the paper, researchers from Johns Hopkins University and elsewhere found a natural long-form transactivating CRISPR RNA (tracr-L) in Streptococcus pyogenes that functions to downregulate its endogenous CRISPR-Cas9 system.

A CRISPR-Cas9 system has two components: the Cas9 guide RNA that directs the system to edit a particular gene, and the CRISPR “scissors” that make the cut. The authors found that tracr-L redirects Cas9 in S. pyogenes to repress its own promoter, functionally decreasing the amount of cutting that occurs. In bacteria with unaltered tracr-L, levels of CRISPR-related genes were low. But altering the tracr-L with genetic engineering to make it function more like a guide RNA increased CRISPR-Cas9 cuts. And removing tracr-L altogether greatly increased CRISPR-Cas9 expression.

The authors suggest natural tracr-L regulates autoimmunity. In bacteria without tracr-L to repress CRISPR-Cas9 expression, immunity to bacteriophage infection increased 3,000-fold. But in cells not challenged by bacteriophages, the number of bacteria dropped by a third in one day, compared with wild-type bacteria.

Joshua Modell, an author on the paper and assistant professor of molecular biology and genetics at the Johns Hopkins University School of Medicine, said the lab will next explore how tracr-L is regulated in bacteria, which could lead to a regulatable guide RDA – “one this is silenced until you can turn its activity on or up.”

At least five companies are using CRISPR-Cas9 systems to develop therapeutics: Caribou Biosciences, Intellia Therapeutics, CRISPR Therapeutics, ERS Genomics and Editas Medicine. In particular, Editas is known to be exploring tunable CRISPR-Cas9 systems for therapies has developed self-inactivating Cas9 adeno-associated virus vectors. And in 2019, Caribou co-founder and CRISPR pioneer Jennifer Doudna co-authored a Nature Communications paper describing a system of activating and deactivating single guide RNAs using small-molecule ligands.