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Grybauskas, A.

Publications and source records attributed to Grybauskas, A..

2 recordsLinked to original sources

Activation of SPARDA defense system by filament assembly reveals beta-relay signaling mechanism widespread in prokaryotic Argonautes

Present in all three domains of life, Argonaute (Ago) proteins use short oligonucleotides as guides to recognize complementary nucleic acid targets and are involved in RNA silencing (eukaryotes) or host defense against invading DNA (prokaryotes). Here, we show that the SPARDA (short prokaryotic Argonaute, DNase associated) system functions in anti-plasmid defense. Upon detection of invading plasmid DNA, it degrades both the invader and host DNA, inducing cell death and preventing further spread of the invader. Upon activation, the recognition signal of the bound guide/target duplex is relayed to other functional SPARDA sites through a structural region, which we termed beta-relay. The associated dramatic conformational changes trigger the formation of a filament, in which the DREN nuclease domains form tetramers poised to cleave dsDNA. Furthermore, we identified the presence of beta-relay in all pAgo clades, providing new insights into the structural mechanisms of pAgo proteins.

biochemistry↗

Off-target detection of CRISPR-Cas9 nuclease in vitro with CROFT-Seq

Programmable CRISPR-Cas9 nucleases have become invaluable tools for genome editing. However, off-target cleavage by these nucleases could lead to unintended changes in the edited genome. Detection of off-target sites is critical to make genome editing technology safe and predictable. Although current in vitro methods for off-target detection can identify these sites, they are time-consuming, complex, and relatively costly. Here, we present CROFT-Seq (CRISPR nuclease off-target detection by sequencing), a sensitive, rapid, and cost-effective assay for the genome-wide detection of Cas9 off-target sites in vitro. CROFT-Seq performs comparably to the common currently used in vitro methods and serves as a valuable and efficient tool for the rapid assessment of genome-editing nuclease specificity. Notably, a high proportion of the top-ranked off-targets identified by CROFT-Seq were validated in cells, highlighting its effectiveness as a predictor of off-target sites.

biochemistry↗