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

文献类型： 外文期刊

作者： Zou, Gen 1 ; Xiao, Meili 1 ; Chai, Shunxing 1 ; Zhu, Zhihua 1 ; Wang, Ying 2 ; Zhou, Zhihua 1 ;

作者机构： 1.Chinese Acad Sci, CAS Ctr Excellence Mol Plant Sci, Inst Plant Physiol & Ecol, CAS Key Lab Synthet Biol, Fenglin Rd 300, Shanghai 200032, Peoples R China

2.Shanghai Acad Agr Sci, Inst Edible Fungi, Shanghai Key Lab Agr Genet & Breeding, 1000 Jinqi Rd, Shanghai 201403, Peoples R China

3.Univ Chinese Acad Sci, Beijing 100049, Peoples R China

期刊名称：MICROBIAL BIOTECHNOLOGY （ 影响因子：5.813； 五年影响因子：6.559 ）

ISSN： 1751-7915

年卷期：

页码：

收录情况： SCI

摘要： 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 assembledin vitrohas 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% inTrichoderma reeseiandCordyceps 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) inT. 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) fromAspergillus 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 onin vivoexpression 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.

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