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IgorG. Sinelnikov, Valeriy Yu. Kislitsin, Andrey M. Chulkin, Andrey A. Shaplin, Aleksandra M. Rozhkova
Influence of ku70 gene knockout on transformation
frequency of Penicillium Verruculosum mycelial fungi
Abstract
Annotation. To
increase the frequency of homologous recombination (HR) during the
transformation of the industrial strain Penicillium verruculosum 221-151
(VKM F-3972D), the ku70 gene encoding the Ku70, which binds at the sites
of double-stranded DNA breaks and is involved in the repair process by the
non-homologous end joint (NHEJ), was knocked out by the CRISPR/CAS9 method.
Presumably, the new host strain, P. verruculosumΔniaDΔku70, should have had an increased
frequency of homologous recombination during transformation in comparison with
the host strain P. verruculosum ΔniaD due to the integrative
insertion of the expression cassette only by the HR mechanism. The pep1
gene encoding homologous aspartate protease was chosen as a marker. However, it
was shown that the knockout of the ku70 gene led to a dramatic decrease
in the frequency of co-transformation in the P. verruculosumΔniaDΔku70 strain compared to the P.
verruculosum ΔniaD strain at the same load of exogenous DNA (3
μg). The number of copies of the pep1 gene in recombinant strains
of the P. verruculosum Pep1 (with a native Ku70) series ranged from 3 to
28 copies, which indicated the predominance of the non-homologous recombination
mechanism.
Key words:
CRISPR/CAS9, ku70, homologous recombination, non-homologous end joint
Copyright (C) Chemistry Dept., Moscow State University, 2002
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