RESUMO
CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable genes. Recently developed Cas9 variants have been engineered with relaxed PAM requirements, including SpG-Cas9 (SpG) and the nearly PAM-less SpRY-Cas9 (SpRY). However, the molecular mechanisms of how SpRY recognizes all potential PAM sequences remains unclear. Here, we combine structural and biochemical approaches to determine how SpRY interrogates DNA and recognizes target sites. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region, which facilitates tight binding to off-target DNA sequences. Nuclease activation occurs ~1000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Experiments with SpG position it as an intermediate enzyme between Cas9 and SpRY. Our findings shed light on the molecular mechanisms of PAMless genome editing.
Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , DNA , Edição de Genes , Streptococcus pyogenes , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Edição de Genes/métodos , DNA/metabolismo , DNA/genética , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , RNA Guia de Sistemas CRISPR-Cas/genéticaRESUMO
A modern apparatus for performing flash chromatography using commercially available, prepacked silica cartridges has been developed. The key advantage of this system, when compared to traditional flash chromatography, is its use of commercially available silica cartridges, which obviates the need for students to handle silica gel. The apparatus has been tested for its ability to perform separations that are commonly found in organic chemistry teaching laboratories, and a laboratory module that combines the techniques of thin-layer chromatography, gas chromatography, and flash chromatography is described. The performance of this new chromatography apparatus was comparable to a traditional flash chromatography column.