MersV1, Main, Exploration, bibRecord, 000231

DNA interference by a mesophilic Argonaute protein, CbcAgo

Identifieur interne : 000231 ( Main/Exploration ); précédent : 000230; suivant : 000232

DNA interference by a mesophilic Argonaute protein, CbcAgo

Auteurs : Nieves García-Quintans [Espagne] ; Laurie Bowden [Espagne] ; José Berenguer [Espagne] ; Mario Mencía [Espagne]

Source :

F1000Research [ 2046-1402 ] ; 2020.

RBID : PMC:6961421

Abstract

Background: The search for putative enzymes that can facilitate gene editing has recently focused its attention on Argonaute proteins from prokaryotes (pAgos). Though they are structural homologues of human Argonaute protein, which uses RNA guides to interfere with RNA targets, pAgos use ssDNA guides to identify and, in many cases, cut a complementary DNA target. Thermophilic pAgos from Thermus thermophilus, Pyrococcus furiosus and Methanocaldococcus jasmanii have been identified and thoroughly studied, but their thermoactivity makes them of little use in mesophilic systems such as mammalian cells.

Methods: Here we search for and identify CbcAgo, a prokaryotic Argonaute protein from a mesophilic bacterium, and characterize in vitro its DNA interference activity.

Results: CbcAgo efficiently uses 5’P-ssDNA guides as small as 11-mers to cut ssDNA targets, requires divalent cations (preferentially, Mn ²⁺) and has a maximum activity between 37 and 42 °C, remaining active up to 55 °C. Nicking activity on supercoiled dsDNA was shown. However, no efficient double-strand breaking activity could be demonstrated.

Conclusions: CbcAgo can use gDNA guides as small as 11 nucleotides long to cut complementary ssDNA targets at 37ºC, making it a promising starting point for the development of new gene editing tools for mammalian cells.

Url:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961421

DOI: 10.12688/f1000research.18445.2
PubMed: 32055395
PubMed Central: 6961421

Affiliations:

Le document en format XML

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<author><name sortKey="Mencia, Mario" sort="Mencia, Mario" uniqKey="Mencia M" first="Mario" last="Mencía">Mario Mencía</name>
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">DNA interference by a mesophilic Argonaute protein, CbcAgo</title>
<author><name sortKey="Garcia Quintans, Nieves" sort="Garcia Quintans, Nieves" uniqKey="Garcia Quintans N" first="Nieves" last="García-Quintans">Nieves García-Quintans</name>
<affiliation wicri:level="2"><nlm:aff id="a1">Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Madrid, 28049, Spain</nlm:aff>
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<placeName><region nuts="2" type="communauté">Communauté de Madrid</region>
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<author><name sortKey="Mencia, Mario" sort="Mencia, Mario" uniqKey="Mencia M" first="Mario" last="Mencía">Mario Mencía</name>
<affiliation wicri:level="2"><nlm:aff id="a1">Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Madrid, 28049, Spain</nlm:aff>
<country xml:lang="fr">Espagne</country>
<wicri:regionArea>Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Madrid, 28049</wicri:regionArea>
<placeName><region nuts="2" type="communauté">Communauté de Madrid</region>
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<series><title level="j">F1000Research</title>
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<front><div type="abstract" xml:lang="en"><p><bold>Background</bold>: The search for putative enzymes that can facilitate gene editing has recently focused its attention on Argonaute proteins from prokaryotes (pAgos). Though they are structural homologues of human Argonaute protein, which uses RNA guides to interfere with RNA targets, pAgos use ssDNA guides to identify and, in many cases, cut a complementary DNA target. Thermophilic pAgos from
<italic>Thermus thermophilus</italic>,
<italic>Pyrococcus furiosus</italic> and
<italic>Methanocaldococcus jasmanii</italic>
 have been identified and thoroughly studied, but their thermoactivity makes them of little use in mesophilic systems such as mammalian cells.</p>
<p><bold>Methods</bold>: Here we search for and identify CbcAgo, a prokaryotic Argonaute protein from a mesophilic bacterium, and characterize
<italic>in vitro</italic>
 its DNA interference activity.</p>
<p><bold>Results</bold>: CbcAgo efficiently uses 5’P-ssDNA guides as small as 11-mers to cut ssDNA targets, requires divalent cations (preferentially, Mn
<sup>2+</sup>
) and has a maximum activity between 37 and 42 °C, remaining active up to 55 °C. Nicking activity on supercoiled dsDNA was shown. However, no efficient double-strand breaking activity could be demonstrated.</p>
<p><bold>Conclusions</bold>
: CbcAgo can use gDNA guides as small as 11 nucleotides long to cut complementary ssDNA targets at 37ºC, making it a promising starting point for the development of new gene editing tools  for mammalian cells.</p>
</div>
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Serveur d'exploration MERS

DNA interference by a mesophilic Argonaute protein, CbcAgo

DNA interference by a mesophilic Argonaute protein, CbcAgo

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Abstract

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