Efficient genome editing in filamentous fungi via an improved CRISPR‐Cas9 ribonucleoprotein method facilitated by chemical reagents

Summary DNA double‐strand break (DSB) repair induced by the RNA‐programmed nuclease Cas9 has become a popular method for genome editing. Direct genome editing via Cas9‐CRISPR gRNA (guide RNA) ribonucleoprotein (RNP) complexes assembled in vitro has also been successful in some fungi. However, the ef...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Gen Zou, Meili Xiao, Shunxing Chai, Zhihua Zhu, Ying Wang, Zhihua Zhou
Formato:	article
Lenguaje:	EN
Publicado:	Wiley 2021
Materias:	Biotechnology TP248.13-248.65
Acceso en línea:	https://doaj.org/article/309331d0df37402ba7234eca6db112d2
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:309331d0df37402ba7234eca6db112d2
record_format	dspace
spelling	oai:doaj.org-article:309331d0df37402ba7234eca6db112d22021-11-18T15:39:52ZEfficient genome editing in filamentous fungi via an improved CRISPR‐Cas9 ribonucleoprotein method facilitated by chemical reagents1751-791510.1111/1751-7915.13652https://doaj.org/article/309331d0df37402ba7234eca6db112d22021-11-01T00:00:00Zhttps://doi.org/10.1111/1751-7915.13652https://doaj.org/toc/1751-7915Summary DNA double‐strand break (DSB) repair induced by the RNA‐programmed nuclease Cas9 has become a popular method for genome editing. Direct genome editing via Cas9‐CRISPR gRNA (guide RNA) ribonucleoprotein (RNP) complexes assembled in vitro has also been successful in some fungi. However, the efficiency of direct RNP transformation into fungal protoplasts is currently too low. Here, we report an optimized genome editing approach for filamentous fungi based on RNPs facilitated by adding chemical reagents. We increased the transformation efficiency of RNPs significantly by adding Triton X‐100 and prolonging the incubation time, and the editing efficiency reached 100% in Trichoderma reesei and Cordyceps militaris. The optimized RNP‐based method also achieved efficient (56.52%) homologous recombination integration with short homology arms (20 bp) and gene disruption (7.37%) that excludes any foreign DNA (selection marker) in T. reesei. In particular, after adding reagents related to mitosis and cell division, the further optimized protocol showed an increased ratio of edited homokaryotic transformants (from 0% to 40.0% for inositol and 71.43% for benomyl) from Aspergillus oryzae, which contains multinucleate spores and protoplasts. Furthermore, the multi‐target engineering efficiency of the optimized RNP transformation method was similar to those of methods based on in vivo expression of Cas9. This newly established genome editing system based on RNPs may be widely applicable to construction of genome‐edited fungi for the food and medical industries, and has good prospects for commercialization.Gen ZouMeili XiaoShunxing ChaiZhihua ZhuYing WangZhihua ZhouWileyarticleBiotechnologyTP248.13-248.65ENMicrobial Biotechnology, Vol 14, Iss 6, Pp 2343-2355 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Biotechnology TP248.13-248.65
spellingShingle	Biotechnology TP248.13-248.65 Gen Zou Meili Xiao Shunxing Chai Zhihua Zhu Ying Wang Zhihua Zhou Efficient genome editing in filamentous fungi via an improved CRISPR‐Cas9 ribonucleoprotein method facilitated by chemical reagents
description	Summary DNA double‐strand break (DSB) repair induced by the RNA‐programmed nuclease Cas9 has become a popular method for genome editing. Direct genome editing via Cas9‐CRISPR gRNA (guide RNA) ribonucleoprotein (RNP) complexes assembled in vitro has also been successful in some fungi. However, the efficiency of direct RNP transformation into fungal protoplasts is currently too low. Here, we report an optimized genome editing approach for filamentous fungi based on RNPs facilitated by adding chemical reagents. We increased the transformation efficiency of RNPs significantly by adding Triton X‐100 and prolonging the incubation time, and the editing efficiency reached 100% in Trichoderma reesei and Cordyceps militaris. The optimized RNP‐based method also achieved efficient (56.52%) homologous recombination integration with short homology arms (20 bp) and gene disruption (7.37%) that excludes any foreign DNA (selection marker) in T. reesei. In particular, after adding reagents related to mitosis and cell division, the further optimized protocol showed an increased ratio of edited homokaryotic transformants (from 0% to 40.0% for inositol and 71.43% for benomyl) from Aspergillus oryzae, which contains multinucleate spores and protoplasts. Furthermore, the multi‐target engineering efficiency of the optimized RNP transformation method was similar to those of methods based on in vivo expression of Cas9. This newly established genome editing system based on RNPs may be widely applicable to construction of genome‐edited fungi for the food and medical industries, and has good prospects for commercialization.
format	article
author	Gen Zou Meili Xiao Shunxing Chai Zhihua Zhu Ying Wang Zhihua Zhou
author_facet	Gen Zou Meili Xiao Shunxing Chai Zhihua Zhu Ying Wang Zhihua Zhou
author_sort	Gen Zou
title	Efficient genome editing in filamentous fungi via an improved CRISPR‐Cas9 ribonucleoprotein method facilitated by chemical reagents
title_short	Efficient genome editing in filamentous fungi via an improved CRISPR‐Cas9 ribonucleoprotein method facilitated by chemical reagents
title_full	Efficient genome editing in filamentous fungi via an improved CRISPR‐Cas9 ribonucleoprotein method facilitated by chemical reagents
title_fullStr	Efficient genome editing in filamentous fungi via an improved CRISPR‐Cas9 ribonucleoprotein method facilitated by chemical reagents
title_full_unstemmed	Efficient genome editing in filamentous fungi via an improved CRISPR‐Cas9 ribonucleoprotein method facilitated by chemical reagents
title_sort	efficient genome editing in filamentous fungi via an improved crispr‐cas9 ribonucleoprotein method facilitated by chemical reagents
publisher	Wiley
publishDate	2021
url	https://doaj.org/article/309331d0df37402ba7234eca6db112d2
work_keys_str_mv	AT genzou efficientgenomeeditinginfilamentousfungiviaanimprovedcrisprcas9ribonucleoproteinmethodfacilitatedbychemicalreagents AT meilixiao efficientgenomeeditinginfilamentousfungiviaanimprovedcrisprcas9ribonucleoproteinmethodfacilitatedbychemicalreagents AT shunxingchai efficientgenomeeditinginfilamentousfungiviaanimprovedcrisprcas9ribonucleoproteinmethodfacilitatedbychemicalreagents AT zhihuazhu efficientgenomeeditinginfilamentousfungiviaanimprovedcrisprcas9ribonucleoproteinmethodfacilitatedbychemicalreagents AT yingwang efficientgenomeeditinginfilamentousfungiviaanimprovedcrisprcas9ribonucleoproteinmethodfacilitatedbychemicalreagents AT zhihuazhou efficientgenomeeditinginfilamentousfungiviaanimprovedcrisprcas9ribonucleoproteinmethodfacilitatedbychemicalreagents
_version_	1718420776681472000

Efficient genome editing in filamentous fungi via an improved CRISPR‐Cas9 ribonucleoprotein method facilitated by chemical reagents

Ejemplares similares