التفاصيل البيبلوغرافية
| العنوان: |
C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector |
| المؤلفون: |
Abudayyeh, Omar O., Gootenberg, Jonathan S., Konermann, Silvana, Joung, Julia, Slaymaker, Ian M., Cox, David B. T., Shmakov, Sergey, Makarova, Kira S., Semenova, Ekaterina, Minakhin, Leonid, Severinov, Konstantin, Regev, Aviv, Lander, Eric S., Koonin, Eugene V., Zhang, Feng |
| المساهمون: |
National Institute of General Medical Sciences, U.S. Department of Health and Human Services, NIH, Russian Science Foundation, Skoltech, New York Stem Cell Foundation, National Institute of Mental Health, NSF, New York Stem Cell, Simons, Paul G. Allen Family, Vallee Foundation |
| المصدر: |
Science ; volume 353, issue 6299 ; ISSN 0036-8075 1095-9203 |
| بيانات النشر: |
American Association for the Advancement of Science (AAAS) |
| سنة النشر: |
2016 |
| الوصف: |
INTRODUCTION Almost all archaea and about half of bacteria possess clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated genes (Cas) adaptive immune systems, which protect microbes against viruses and other foreign DNA. All functionally characterized CRISPR systems have been reported to target DNA, with some multicomponent type III systems also targeting RNA. The putative class 2 type VI system, which has not been functionally characterized, encompasses the single-effector protein C2c2, which contains two Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains commonly associated with ribonucleases (RNases), suggesting RNA-guided RNA-targeting function. RATIONALE Existing studies have only established a role for RNA interference, in addition to DNA interference, in the multicomponent type III-A and III-B systems. We investigated the possibility of C2c2-mediated RNA inference by heterologously expressing C2c2 locus from Leptotrichia shahii (LshC2c2) in the model system Escherichia coli. The ability of LshC2c2 to protect against MS2 single-stranded RNA (ssRNA) phage infection was assessed by using every possible spacer sequence against the phage genome. We next developed protocols to reconstitute purified recombinant LshC2c2 protein and test its biochemical activity when incubated with its mature CRISPR RNA (crRNA) and target ssRNA. We systematically evaluated the parameters necessary for cleavage. Last, to demonstrate the potential utility of the LshC2c2 complex for RNA targeting in living bacterial cells, we guided it to knockdown red fluorescent protein (RFP) mRNA in vivo. RESULTS This work demonstrates the RNA-guided RNase activity of the putative type VI CRISPR-effector LshC2c2. Heterologously expressed C2c2 can protect E. coli from MS2 phage, and by screening against the MS2 genome, we identified a H (non-G) protospacer flanking site (PFS) following the RNA target site, which was confirmed by targeting a complementary sequence in the β-lactamase transcript followed ... |
| نوع الوثيقة: |
article in journal/newspaper |
| اللغة: |
English |
| DOI: |
10.1126/science.aaf5573 |
| الإتاحة: |
https://doi.org/10.1126/science.aaf5573Test |
| رقم الانضمام: |
edsbas.E8C0B0BC |
| قاعدة البيانات: |
BASE |
