Identification of the HECT E3 ligase UBR5 as a regulator of MYC degradation using a CRISPR/Cas9 screen.

MYC oncoprotein is a multifunctional transcription factor that regulates the expression of a large number of genes involved in cellular growth, proliferation and metabolism. Altered MYC protein level lead to cellular transformation and tumorigenesis. MYC is deregulated in > 50% of human cancers, rendering it an attractive drug target.

Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6.

Bacterial and archaeal CRISPR-Cas systems provide RNA-guided immunity against genetic invaders such as bacteriophages and plasmids. Upon target RNA recognition, type III CRISPR-Cas systems produce cyclic-oligoadenylate second messengers that activate downstream effectors, including Csm6 ribonucleases, via their CARF domains. Here, we show that Enteroccocus italicus Csm6 (EiCsm6) degrades its cognate cyclic hexa-AMP (cA6) activator, and report the crystal structure of EiCsm6 bound to a cA6 mimic.

Specialized Weaponry: How a Type III-A CRISPR-Cas System Excels at Combating Phages.

CRISPR-Cas-mediated defense against phage invaders usually requires recognition of short sequences, termed protospacer-adjacent motifs (PAMs), in phage DNA. In this issue of Cell Host & Microbe, Pyenson et al. (2017) show that the lack of a PAM requirement in some CRISPR-Cas systems prevents interference evasion and facilitates phage extinction.

Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers.

In many prokaryotes, type III clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) systems detect and degrade invasive genetic elements by an RNA-guided, RNA-targeting multisubunit interference complex. The CRISPR-associated protein Csm6 additionally contributes to interference by functioning as a standalone RNase that degrades invader RNA transcripts, but the mechanism linking invader sensing to Csm6 activity is not understood. Here we show that Csm6 proteins are activated through a second messenger generated by the type III interference complex.

Structural basis for the endoribonuclease activity of the type III-A CRISPR-associated protein Csm6.

Prokaryotic CRISPR-Cas systems provide an RNA-guided mechanism for genome defense against mobile genetic elements such as viruses and plasmids. In type III-A CRISPR-Cas systems, the RNA-guided multisubunit Csm effector complex targets both single-stranded RNAs and double-stranded DNAs. In addition to the Csm complex, efficient anti-plasmid immunity mediated by type III-A systems also requires the CRISPR-associated protein Csm6.

In Vitro Reconstitution and Crystallization of Cas9 Endonuclease Bound to a Guide RNA and a DNA Target.

The programmable RNA-guided DNA cleavage activity of the bacterial CRISPR-associated endonuclease Cas9 is the basis of genome editing applications in numerous model organisms and cell types. In a binary complex with a dual crRNA:tracrRNA guide or single-molecule guide RNA, Cas9 targets double-stranded DNAs harboring sequences complementary to a 20-nucleotide segment in the guide RNA. Recent structural studies of the enzyme have uncovered the molecular mechanism of RNA-guided DNA recognition.

Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease.

The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA.

Evolution of CRISPR RNA recognition and processing by Cas6 endonucleases.

In many bacteria and archaea, small RNAs derived from clustered regularly interspaced short palindromic repeats (CRISPRs) associate with CRISPR-associated (Cas) proteins to target foreign DNA for destruction. In Type I and III CRISPR/Cas systems, the Cas6 family of endoribonucleases generates functional CRISPR-derived RNAs by site-specific cleavage of repeat sequences in precursor transcripts. CRISPR repeats differ widely in both sequence and structure, with varying propensity to form hairpin folds immediately preceding the cleavage site.