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Genetic Engineering Publications - GEG Tech top picks
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BigField GEG Tech's insight:
The authors of this study previously identified he first examples of proteins that inhibit a CRISPR–Cas system. Here they performed biochemical and in vivo investigations of three of these anti-CRISPR proteins, and show that each inhibits CRISPR–Cas activity through a distinct mechanism. The diverse sequences and mechanisms of action of these anti-CRISPR proteins imply an independent evolution, and foreshadow the existence of other means by which proteins may alter CRISPR–Cas function. 
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BigField GEG Tech's insight:
Here, the scientists describe the application of an inducible, RNA-guided, nuclease-deficient Cas9-KRAB system to silence target gene expression in human embryonic stem cells, via KRAB repression at the promoter region. This chapter outlines a detailed protocol for generation of a stable human embryonic stem cell line containing both Sp-dCas9-KRAB and sgRNA, followed by inducible expression of Sp-dCas9-KRAB to analyze functional effects of dCas9-KRAB at target loci in human embryonic stem cells.
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Recently, two anti-CRISPR proteins AcrIIA2 and AcrIIA4 have been identified, both of which potently inhibit Cas9 (SpyCas9) and LmoCas9 activity in bacteria and human cells. However, the mechanism of Cas9 inhibition remains unknown. Here the scientists report a crystal structure of SpyCas9 in complex with a single guide RNA (sgRNA) and AcrIIA4. Their results reveal the mechanism of SpyCas9 inhibition by AcrIIA4, laying a structural basis for developing "off-switch" tools of SpyCas9 to avoid unwanted genome edits within cells and tissues.