Broad Targeting Specificity during Bacterial Type III CRISPR-Cas Immunity Constrains Viral Escape Nora C. Pyenson, Kaitlyn Gayvert, Andrew Varble, Olivier Elemento, and Luciano A. Marraffini Cell Host Microbe. 2017 Sep 13;22(3):343-353.e3. Speaker: Ke-Ying Hsieh (謝可盈) Time:13:00~14:00, March,14th, 2017 Commentator: Dr. Jenn-Wei Chen (陳振暐老師) Place: Room 601 Abstract: CRISPR-Cas system is the basis of adaptive immunity in prokaryotes. It consists of CRISPR (clustered regularly interspaced short palindromic repeats) loci and many associated genes (cas).1 This system will capture short foreign DNA during virus or plasmid invasion and degrade them.2 There are different ways of targeting depending on different types of CRISPR-Cas immunity. Type Ⅰ and type Ⅱ CRISPR-Cas targeting require a short conserved sequence named PAM (protospacer adjacent motif) flanking the targeting sequence (also known as protospacer) and 6-8 nucleotides of protospacer(also known as seed) fully complementary to the recognition sequence (also known as spacer). Under these strict targeting rules, a single-nucleotide mutation in the PAM or seed can abrogate type Ⅰ and type Ⅱ CRISPR-Cas immunity. On the other hand, type Ⅲ CRISPR-Cas immunity has more flexible targeting rules. It has been shown that this system can tolerate different mutations in the protospacer.3 In this paper, the authors searched for the presence of PAM and seed sequence in type Ⅲ CRISPR-Cas immunity. They find PAM or seed sequence were not required for targeting and single-mutation in PAM or seed can still be targeted by type Ⅲ CRISPR-Cas immunity. Only mutations that make homology flanking the region of prototospacer can prevent targeting. Also the authors demonstrated that phage escaping from type Ⅱ CRISPR-Cas immunity result in mutation in the protospacer, but type Ⅲ CRISPR-Cas immunity result in complete target sequence deletion from the viral genome. Moreover, the authors designed type Ⅲ CRISPR-Cas spacer that targets on viral essential gene which encodes phage major capsid protein and find extinction of the phage. Finally, the authors use demonstrated that type Ⅲ CRISPR-Cas immunity can cause phage extinction by only one spacer. To sum up, the authors have showed us type Ⅲ CRISPR-Cas system is a robust immunity system with broad targeting specificity able to prevent emergence of mutant phage effectively. References: 1. Brouns SJ et al. 2009 CRISPR-based adaptive and heritable immunity in prokaryotes. Trends Biochem Sci. 34(8):401-7. 2. Sorek R et al. 2016 CRISPR-Cas adaptation: insights into the mechanism of action. Nat Rev Microbiol. 14(2):67-76. 3. Goldberg et al. 2014 Conditional tolerance of temperate phages via transcription-dependent CRISPR-Cas targeting. Nature 514, 633–637.