Home
Resource
CRISPR-Cas adaptive Immunity in Leptotrichia shahii Type VI-A system
PDF
PDF format is widely accepted and good for printing.
Plug-in required
PDF-1(2.47 MB)
Citation & Export
View Usage Statistics
Staff View

Citation & Export
Hide

Simple citation

Jain, Ishita. CRISPR-Cas adaptive Immunity in Leptotrichia shahii Type VI-A system. Retrieved from https://doi.org/doi:10.7282/t3-dr5t-dx93

Export

Click here for information about Citation Management Tools at Rutgers.

Statistics
Hide

Description

TitleCRISPR-Cas adaptive Immunity in Leptotrichia shahii Type VI-A system
NameJain, Ishita (author); Severinov, Konstantin (chair); Zylstra, Gerben (member); Chikindas, Michael (member); Meeske, Alexander (member); Rutgers University; School of Graduate Studies
Date Created2022
Other Date2022-01 (degree)
SubjectMolecular biology, Microbiology, Biotechnology, Cas13, CRISPR Cas, Leptotrichia shahii, RNA targeting, Type VI
Extent113 pages : illustrations
DescriptionBacteria and phages are constantly battling an evolutionary arms race where they developed several defense strategies to outcompete each other. Prokaryotes utilize CRISPR-Cas systems, an adaptive immune mechanism to protect themselves by targeting phage genomes in a sequence specific manner. Type VI systems are a unique subtype of Class 2 CRISPR-Cas systems which provide host immunity by exclusively targeting RNA. The Type VI nuclease Cas13, gets activated upon target RNA recognition and causes ‘collateral’ RNA cleavage in trans, which leads to bacterial cell dormancy and protection of the host cells. However, due to a lack of in vivo studies, the mechanistic details of the RNA-targeting activity of Type VI systems remains unclear and raises questions about its role as a prokaryotic defense system.

In this work, we conducted in vivo and in vitro analyses to show that Cas13a effector from Leptotrichia shahii (Lsh) is a specific RNA-targeting nuclease which does not exhibit promiscuous indiscriminate RNase activity as previously suggested. We show that tRNA anticodon loops are true cleavage substrates for target-activated LshCas13a and these cleavages are preferentially made in uridine rich residues.

On the other hand, spacer acquisition is an important process in making CRISPR-Cas systems ‘adaptive’ in nature, where the CRISPR array can store memories of past phage infections and pass onto bacterial progeny. Since the Type VI-A system targets RNA and has no known associated reverse transcriptases, it is important to understand how this system acquires new spacers.

Here, we developed an oligo adaptation assay to test spacer integration in the Type VI-A CRISPR array. We showed that RNA-targeting LshCas13a systems can acquire double-stranded DNA oligos as spacers in the CRISPR array utilizing the Cas1 and Cas2 adaptation proteins. We also demonstrated that RNA oligos are not acquired by the Lsh Type VI-A CRISPR systems.

In light of these results, we also presented a model of Type VI-A CRISPR-Cas adaptive immunity to describe its function as an adaptive prokaryotic defense system.
NotePh.D.
NoteIncludes bibliographical references
Genretheses
Persistent URLhttps://doi.org/doi:10.7282/t3-dr5t-dx93
LanguageEnglish
CollectionSchool of Graduate Studies Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
Version 8.5.5
Rutgers University Libraries - Copyright ©2024