Hinting at a new approach to regulating gene editing in mammalian cells, a study by Jiyung Shin, Jennifer Doudna and colleagues reports that an inhibitor protein from a Listeria bacteriophage can block the Cas9 component of CRISPR-Cas9 from interacting with DNA. Critically, adding this anti-CRISPR protein, known as AcrIIA4, to a human leukemia cell hours after CRISPR-Cas9 was introduced allowed CRISPR-Cas9 to still cut target DNA, while reducing off-target cuts. Insights from this work suggest that AcrIIA4, and other natural inhibitor proteins, could help refine gene editing attempts based on CRIPSR and Cas enzymes, processes which have lacked a controllable "off-switch" to prevent undesired gene changes, to date. While scientists recognize potential applications for Cas9 inhibitor proteins in gene editing of mammalian cells, the mechanisms underlying such processes have not fully been established.
Here, in a structural analysis, Jiyung Shin et al. first examined how the natural inhibitor protein AcrIIA4 interacts with the Cas9 enzyme. They found that AcrIIA4 binds to the enzyme in the "pocket" that DNA would otherwise fit into, blocking the enzyme from attaching to target DNA and cutting it. Studying human cells revealed that if these cells were treated with the inhibitor protein before gene editing began, the CRIPSR-Cas complex was largely prevented from cutting the DNA at any location. Results also showed that about half of CRISPR-Cas9's on-target editing of DNA (in which the system correctly cuts the genes scientists hope to edit) happened within 6 hours of the system being introduced. Some previous research has suggested that the complex may sit on off-target sites for a while without cutting them. Capitalizing on this timeline, Shin et al. introduced a CRISPR-Cas9 complex directed to cut two genes, including the gene responsible for sickle cell disease, into human leukemia cells, and then added AcrIIA4 6 hours later. Results showed adding AcrIIA4 at the right time prevented cutting at the wrong sites while still allowing time for cutting at the right sites.
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