A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity

  title={A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity},
  author={Martin Jinek and Krzysztof Chylinski and Ines Fonfara and Michael H. Hauer and Jennifer A. Doudna and E. Charpentier},
  pages={816 - 821}
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. [] Key Result At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand.
Mechanism and engineering of CRISPR-associated endonucleases
It is revealed that Cas9 can be programmed to target single-stranded RNA substrates for both high-affinity binding and site- specific cleavage using PAM-presenting oligonucleotides and that RNA targeting by Cas9 has the potential to transform the study of RNA function, much as site-specific DNA targeting has revolutionized genetic and genomic research.
RNA-Guided Human Genome Engineering via Cas9
The type II bacterial CRISPR system is engineer to function with custom guide RNA (gRNA) in human cells to establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3
Atomic resolution structures of a full-length Cas3 are shown, revealing how Cas3 coordinates binding, ATP-dependent translocation, and nuclease digestion of invader DNA and revealing important mechanistic details on the neutralization of genetic invaders by type I CRISPR-Cas systems.
Functions and Applications of RNA‐Guided CRISPR‐Cas Immune Systems
The inheritable and hypervariable nature of these loci can be used to track the phylogenetic path of an organism and reveal the evolutionary interplay between hosts and their viruses.
Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation
To compare the architectures and domain organization of diverse Cas9 proteins, the atomic structures of Cas9 from Streptococcus pyogenes and Actinomyces naeslundii and AnaCas9 were determined by x-ray crystallography and three-dimensional reconstructions of apo-SpyCas9, SpyCas9:RNA, and SpyCas 9:RNA:DNA were obtained by negative-stain single-particle electron microscopy.
CRISPR-Cas9 Structures and Mechanisms.
This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage and provides a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas 9-based therapies against genetic diseases.
CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference
Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage, and biochemically that base pairing of the Pam region is unnecessary for target binding but critical for Cas3-mediated degradation.
CRISPR‐Cas systems and RNA‐guided interference
A number of studies have shown that CRISPR‐mediated immunity can readily increase the breadth and depth of virus resistance in bacteria and archaea and applications of crRNA‐guided interference are discussed.
Mechanism of foreign DNA recognition by a CRISPR RNA-guided surveillance complex from Pseudomonas aeruginosa
The Type I-F CRISPR-mediated (clustered regularly interspaced short palindromic repeats) adaptive immune system in Pseudomonas aeruginosa consists of two CRISPR loci and six CRISPR-associated (cas)
A Cas9–guide RNA complex preorganized for target DNA recognition
The guide RNA in the CRISPR-Cas immune/editing system is poised to initiate recognition of target DNA and is construe this as convergent evolution of a “seed” mechanism reminiscent of that used by Argonaute proteins during RNA interference in eukaryotes.


Sequence- and Structure-Specific RNA Processing by a CRISPR Endonuclease
The RNA recognition mechanism identified here explains sequence- and structure-specific processing by a large family of CRISPR-specific endoribonucleases.
RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions
It is shown that the Csy proteins (Csy1–4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences.
CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III
CRISPR/Cas systems constitute a widespread class of immunity systems that protect bacteria and archaea against phages and plasmids, and commonly use repeat/spacer-derived short crRNAs to silence
An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3
It is shown that Cse3 type CRISPR-specific endoRNases bind a hairpin structure and residues downstream of the cleavage site within the repetitive segment of cognateCRISPR RNA, providing insight into a catalytically essential RNA recognition mechanism by a large class of CRISpr-related endo RNases.
Structures of the RNA-guided surveillance complex from a bacterial immune system
Cryo-electron microscopy is used to determine the subnanometre structures of Cascade before and after binding to a target sequence and reveal a sea-horse-shaped architecture in which the crRNAs are displayed along a helical arrangement of protein subunits that protect the crRNA from degradation while maintaining its availability for base pairing.
The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA
In vivo evidence is provided that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading toplasmid loss.
Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes.
This work has identified Pyrococcus furiosus Cas6 as a novel endoribonuclease that cleaves CRISPR RNAs within the repeat sequences to release individual invader targeting RNAs.
Recognition and maturation of effector RNAs in a CRISPR interference pathway
High-resolution X-ray structures of Cse3 bound to repeat RNAs model both the pre- and post-cleavage complexes associated with processing thePre-crRNA and establish the molecular basis of a specific CRISPR RNA recognition and suggest the mechanism for generation of effector RNAs responsible for gene silencing.