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Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria
It is demonstrated that the Cas9–crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system introduces in vitro a double-strand break at a specific site in DNA containing a sequence complementary to crRNA, paving the way for engineering of universal programmable RNA-guided DNA endonucleases.
The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli
The results show that active CRISPR/Cas systems can be transferred across distant genera and provide heterologous interference against invasive nucleic acids and can be leveraged to develop strains more robust against phage attack, and safer organisms less likely to uptake and disseminate plasmid-encoded undesirable genetic elements.
Cas3 is a single‐stranded DNA nuclease and ATP‐dependent helicase in the CRISPR/Cas immune system
It is proposed that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade–crRNA complex targeting foreign DNA.
Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes
Single-molecule DNA supercoiling is used to directly observe and quantify the dynamics of torque-dependent R-loop formation and dissociation for both Cascade- and Cas9-based CRISPR-Cas systems and finds that Cascade has higher torque stability than Cas9 by using a conformational locking step.
In vitro reconstitution of Cascade‐mediated CRISPR immunity in Streptococcus thermophilus
It is shown that Cas3 binding to the displaced strand in the R‐loop triggers DNA cleavage, and if ATP is present, Cas3 further degrades DNA in a unidirectional manner, establishing a molecular basis for CRISPR immunity in St‐CRISPR4‐Cas and other Type I systems.
Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements
This work presents a new in vitro method for the simultaneous examination of guide RNA and protospacer adjacent motif (PAM) requirements of Cas9s, and accurately reproduces the canonical PAM preferences for Streptococcus pyogenes, StrePTococcus thermophilus CRISPR3, andCRISPR1.
Molecular mechanisms of CRISPR-mediated microbial immunity
This review focuses on the molecular mechanisms of interference/immunity provided by different CRISPR–Cas systems, which are invader-specific, adaptive, and heritable.
Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome.
Results show that Cas9 proteins with longer or more restrictive PAM requirements provide a safe alternative to SpCas9-based RGNs and hence a valuable option for future human gene therapy applications.
Lactase non-persistence is directed by DNA variation-dependent epigenetic aging
A comprehensive epigenetic study of human and mouse small intestines, using chromosome-wide DNA-modification profiling and targeted bisulfite sequencing, confirmed the importance of these regulatory elements in modulating lactase mRNA levels by using CRISPR–Cas9-induced deletions and enabled a gradual accumulation of epigenetic changes with age.
crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus
The Cas9-crRNA complex of the Streptococcus thermophilus DGCC7710 CRISPR3-Cas system functions as an RNA-guided endonuclease with crRNA-directed target sequence recognition and protein-mediated DNA