Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli

By Ido Yosef, M. Goren, U. Qimron. Nucleic Acids Research, 2012

665 citations recorded by the source corpus.

The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) constitute a recently identified prokaryotic defense mechanism against invading nucleic acids. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called ‘adaptation’, (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Here we describe a robust assay in Escherichia coli to explore the hitherto least-studied process, adaptation. We identify essential genes and DNA elements in the leader sequence and in the array which are essential for the adaptation step. We also provide mechanistic insights on the insertion of the repeat-spacer unit by showing that the first repeat serves as the template for the newly inserted repeat. Taken together, our results elucidate fundamental steps in the adaptation process of the CRISPR/Cas system.

View Paper: https://www.semanticscholar.org/paper/bb7c7b5b0fc21b4f27222b83364e172a0fd07b10

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Citations in the corpus

CRISPR-Cas Systems: RNA-mediated Adaptive Immunity in Bacteria and Archaea (2013, methodology): “…of articles on spacer uptake in the type I-E system of E. coli under genetically manipulated conditions (Datsenko et al. 2012; Swarts et al. 2012; Yosef et al. 2012) and in the type I-A (and type III-B) systems of S. solfataricus using an environmental viral sample (Erdmann and Garrett 2012),…”
Biology and Applications of CRISPR Systems: Harnessing Nature's Toolbox for Genome Engineering. (2016, unknown): “coli in vivo (Datsenko et al., 2012; Swarts et al., 2012; Yosef et al., 2012).”
CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity (2014, background): “Several studies of model CRISPR-Cas systems have also shown adaptive spacer uptake in E. coli type I-E systems (Datsenko et al., 2012; Swarts et al., 2012; Yosef et al., 2012) from plasmid exposure, as well as in a type III system from Sulfolobus solfataricus (Erdmann and Garrett, 2012).”
The Biology of CRISPR-Cas: Backward and Forward. (2018, unknown): “By compromising the selﬁsh, often hostile programs encoded by MGEs, CRISPR-Cas systems protect prokaryotes from succumbing to infection.”
New CRISPR-Cas systems from uncultivated microbes (2016, background): “Strikingly, both CRISPR-CasY and C2c3 were found next to arrays with short spacers and within loci lacking Cas2, a protein considered essential for integrating DNA into the CRISPR array 9,35 .”
Transcriptome engineering with RNA-targeting Type VI-D CRISPR effectors (2018, unknown): “With the exception of the metagenomic AdmCas13d system, Cas13d systems lack the key spacer acquisition protein Cas1 (Yosef et al., 2012) within their CRISPR locus, highlighting the utility of a class 2 CRISPR discovery pipeline without Cas1 or Cas2 gene requirements.”
C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector (2016, unknown): “In all CRISPR-Cas systems characterized to date, Cas1 and Cas2 are exclusively involved in spacer acquisition (Datsenko et al., 2012; Diez-Villasenor et al., 2013; Heler et al., 2015a; Nunez et al., 2014; Nunez et al., 2015; Yosef et al., 2012), suggesting that C2c2 is the sole effector protein whic”
CRISPRDetect: A flexible algorithm to define CRISPR arrays (2016, background): “Insertions, deletions and substitutions can occur in repeats and may be copied into new repeats during spacer acquisition [50].”