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Production and analysis of synthetic Cascade variants
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) is an adaptive immune system of Archaea and Bacteria. It is able to target and destroy foreign genetic material with ribonucleoprotein complexes consisting of CRISPR RNAs (crRNAs) and certain Cas proteins....
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| Format: | Doctoral Thesis |
| Language: | English |
| Published: |
Philipps-Universität Marburg
2020
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| Subjects: | |
| Online Access: | PDF Full Text |
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| Summary: | CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) is an
adaptive immune system of Archaea and Bacteria. It is able to target and destroy foreign genetic
material with ribonucleoprotein complexes consisting of CRISPR RNAs (crRNAs) and certain Cas proteins.
CRISPR-Cas systems are classified in two major classes and multiple types, according to the involved Cas
proteins. In type I systems, a ribonucleoprotein complex called Cascade (CRISPR associated complex for
antiviral defence) scans for invading viral DNA during a recurring infection and binds the sequence
complementary to the incorporated crRNA. After target recognition, the nuclease/helicase Cas3 is
recruited and subsequently destroys the viral DNA in a step termed interfere nce.
Multiple subtypes of type I exist that show differences in the Cascade composition. This work focuses on
a minimal Cascade variant found in Shewanella putrefaciens CN-32. In comparison to the well-studied
type I-E Cascade from Escherichia coli, this complex is missing two proteins usually required for target
recognition, yet it is still able to provide immunity. Recombinant I-Fv Cascade was previously purified
from E. coli and it was possible to modulate the complex by extending or shortening the backbone,
resulting in synthetic variants with altered protein stoichiometry.
In the present study, I-Fv Cascade was further analyzed by in vitro methods. Target binding was
observed and the 3D structure revealed structural variations that replace the missing subunits,
potentially to evade viral anti-CRISPR proteins. The nuclease/helicase of this system, Cas2/3fv, is a fusion
of the Cas3 protein with the interference-unrelated protein Cas2. A standalone Cas3fv was purified
without the Cas2 domain and in vitro cleavage assays showed that Cas3fv degrades both free ssDNA as
well as Cascade-bound substrates. The complete Cas2/3fv protein forms a complex with the protein
Cas1 and was shown to reduce cleave of free ssDNA, potentially as a regulatory mechanism against
unspecific cleavage.
Furthermore, we established a process termed “RNA wrapping”. Synthetic Cascade assemblies can be
created by directing the general RNA-binding ability of the characteristic Cas7fv backbone protein on an
RNA of choice such as reporter gene transcripts. Specific complex formation can be initiated in vivo by
including a repeat sequence from the crRNA upstream a given target sequence and binding of the
Cas5fv protein. The created complexes contain the initial 100 nt of the tagged RNA which can be
isolated afterwards. While incorporated in complexes, RNA is stabilized and protected from degradation
by RNases. Complex formation can be used to silence reporter gene transcripts. Furthermore, we
provided initial indications that the backbone of synthetic complexes can be modified by addition of
reporter proteins. |
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| Physical Description: | 149 Pages |
| DOI: | 10.17192/z2020.0099 |