I-SceI enzyme mediated integration (SEMI) for fast and efficient gene targeting in Trichoderma reesei.

Abstract:

:We previously showed that creation of a double strand DNA break (DSB) by expressing I-SceI in an engineered Trichoderma reesei (Hypocrea jecorina) strain containing a I-SceI recognition site improved transformation and homologous integration efficiencies. In this study, we further improved homologous integration frequencies by combining I-SceI mediated double strand break with disruption of the tku70 gene. The inability of the tku70 mutant to repair a I-SceI mediated DSB via NHEJ was used to force integration of an expression cassette with homologous flanks surrounding the DSB site. Besides expressing I-SceI from a plasmid, we also show that adding I-SceI enzyme during transformation was successful to generate DSBs. The I-SceI enzyme mediated integration, or SEMI, in combination with a Δtku70 mutant has a synergistic effect on homologous recombination efficiencies as 90-100% of the transformants exhibited integration of the expression cassette at the homologous site.

journal_name

J Biotechnol

journal_title

Journal of biotechnology

authors

Ouedraogo JP,Arentshorst M,Nikolaev I,Barends S,Ram AF

doi

10.1016/j.jbiotec.2016.02.012

subject

Has Abstract

pub_date

2016-03-20 00:00:00

pages

25-8

eissn

0168-1656

issn

1873-4863

pii

S0168-1656(16)30057-8

journal_volume

222

pub_type

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