Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex
Abstract Background CRISPR/Cas9 has wide application potentials in a variety of biological species including Trichoderma reesei, a filamentous fungus workhorse for cellulase production. However, expression of Cas9 heterologously in the host cell could be time-consuming and sometimes even troublesome...
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doaj-f7d87169d3304977938f6fe56c5a87e52020-11-25T03:54:40ZengBMCBMC Biotechnology1472-67502019-01-011911710.1186/s12896-018-0498-yFast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complexZhenzhen Hao0Xiaoyun Su1Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural SciencesKey Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural SciencesAbstract Background CRISPR/Cas9 has wide application potentials in a variety of biological species including Trichoderma reesei, a filamentous fungus workhorse for cellulase production. However, expression of Cas9 heterologously in the host cell could be time-consuming and sometimes even troublesome. Results We tested two gene disruption methods in T. reesei using CRISPR/Cas9 in this study. The intracellularly expressed Cas9 led to unexpected off-target gene disruption in T. reesei QM9414, favoring inserting 9- or 12-bp at 70- and 100-bp downstream of the targeted ura5. An alternative method was, therefore, established by assembling Cas9 and gRNA in vitro, followed by transformation of the ribonucleoprotein complex with a plasmid containing the pyr4 marker gene into T. reesei TU-6. When the gRNA targeting cbh1 was used, eight among the twenty seven transformants were found to lose the ability to express CBH1, indicative of successful cbh1 disruption through genome editing. Large DNA fragments including the co-transformed plasmid, chromosomal genes, or a mixture of these nucleotides, were inserted in the disrupted cbh1 locus. Conclusions Direct transformation of Cas9/gRNA complex into the cell is a fast means to disrupt a gene in T. reesei and may find wide applications in strain improvement and functional genomics study.http://link.springer.com/article/10.1186/s12896-018-0498-yCRISPR/Cas9Trichoderma reeseiGene disruptionRibonucleoprotein |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Zhenzhen Hao Xiaoyun Su |
spellingShingle |
Zhenzhen Hao Xiaoyun Su Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex BMC Biotechnology CRISPR/Cas9 Trichoderma reesei Gene disruption Ribonucleoprotein |
author_facet |
Zhenzhen Hao Xiaoyun Su |
author_sort |
Zhenzhen Hao |
title |
Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex |
title_short |
Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex |
title_full |
Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex |
title_fullStr |
Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex |
title_full_unstemmed |
Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex |
title_sort |
fast gene disruption in trichoderma reesei using in vitro assembled cas9/grna complex |
publisher |
BMC |
series |
BMC Biotechnology |
issn |
1472-6750 |
publishDate |
2019-01-01 |
description |
Abstract Background CRISPR/Cas9 has wide application potentials in a variety of biological species including Trichoderma reesei, a filamentous fungus workhorse for cellulase production. However, expression of Cas9 heterologously in the host cell could be time-consuming and sometimes even troublesome. Results We tested two gene disruption methods in T. reesei using CRISPR/Cas9 in this study. The intracellularly expressed Cas9 led to unexpected off-target gene disruption in T. reesei QM9414, favoring inserting 9- or 12-bp at 70- and 100-bp downstream of the targeted ura5. An alternative method was, therefore, established by assembling Cas9 and gRNA in vitro, followed by transformation of the ribonucleoprotein complex with a plasmid containing the pyr4 marker gene into T. reesei TU-6. When the gRNA targeting cbh1 was used, eight among the twenty seven transformants were found to lose the ability to express CBH1, indicative of successful cbh1 disruption through genome editing. Large DNA fragments including the co-transformed plasmid, chromosomal genes, or a mixture of these nucleotides, were inserted in the disrupted cbh1 locus. Conclusions Direct transformation of Cas9/gRNA complex into the cell is a fast means to disrupt a gene in T. reesei and may find wide applications in strain improvement and functional genomics study. |
topic |
CRISPR/Cas9 Trichoderma reesei Gene disruption Ribonucleoprotein |
url |
http://link.springer.com/article/10.1186/s12896-018-0498-y |
work_keys_str_mv |
AT zhenzhenhao fastgenedisruptionintrichodermareeseiusinginvitroassembledcas9grnacomplex AT xiaoyunsu fastgenedisruptionintrichodermareeseiusinginvitroassembledcas9grnacomplex |
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