Cross-cleavage activity of Cas6b in crRNA processing of two different CRISPR-Cas systems in Methanosarcina mazei Gö1

RNA Biology
Lisa NickelRuth A Schmitz

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR) system is a prokaryotic adaptive defense system against foreign nucleic acids. In the methanoarchaeon Methanosarcina mazei Gö1, two types of CRISPR-Cas systems are present (type I-B and type III-C). Both loci encode a Cas6 endonuclease, Cas6b-IB and Cas6b-IIIC, typically responsible for maturation of functional short CRISPR RNAs (crRNAs). To evaluate potential cross cleavage activity, we biochemically characterized both Cas6b proteins regarding their crRNA binding behavior and their ability to process pre-crRNA from the respective CRISPR array in vivo. Maturation of crRNA was studied in the respective single deletion mutants by northern blot and RNA-Seq analysis demonstrating that in vivo primarily Cas6b-IB is responsible for crRNA processing of both CRISPR arrays. Tentative protein level evidence for the translation of both Cas6b proteins under standard growth conditions was detected, arguing for different activities or a potential non-redundant role of Cas6b-IIIC within the cell. Conservation of both Cas6 endonucleases was observed in several other M. mazei isolates, though a wide variety was displayed. In general, repeat and leader sequence conservation re...Continue Reading

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Citations

Mar 21, 2020·Frontiers in Bioengineering and Biotechnology·Yanli ZhengWenfang Peng
Feb 13, 2020·International Journal of Molecular Sciences·Michal BurmistrzAgata Krawczyk-Balska
Dec 9, 2020·Nucleic Acids Research·Alexander MitrofanovRolf Backofen
Apr 17, 2021·International Journal of Biological Macromolecules·Aman Prakash, Manish Kumar

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Methods Mentioned

BETA
size exclusion chromatography
dot blot
gel-filtration
RNA-Seq
PCR
size-exclusion chromatography
dRNA-Seq

Software Mentioned

Integrated Genome Browser
MAFFT
CRISPER
MegaBLAST
fastq
FASTX
Raytest
READemption
AIDA
- identifier

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