CRISPR/Cas technologies constitute essential tools for rapid genome engineering of many organisms, including fungi. The CRISPR/Cas9 system adapted for the industrial fungus Ashbya gossypii enables efficient genome editing for the introduction of deletions, insertions and nucleotide substitutions. However, the Cas9 system is constrained by the existence of a specific 5'-NGG-3' PAM sequence in the target site. Here we present a new CRISPR/Cas system for A. gossypii that expands the molecular toolbox available for microbial engineering of this fungus. The use of Cpf1 nuclease from Lachnospiraceae bacterium allows a T-rich PAM sequence (5'-TTTN-3') to be employed and facilitates implementation of a multiplexing CRISPR/Cpf1 system adapted for A. gossypii. The system has been validated for the introduction of large deletions with five different auxotrophic markers (HIS3, ADE2, TRP1, LEU2 and URA3). The use of both crRNA and dDNA arrays in a multi-CRISPR/Cpf1 system is demonstrated to be an efficient strategy for multiplex gene deletion of up to four genes using a single multi-CRISPR/Cpf1 plasmid. Our results also suggest that the selection of the target sequence may affect significantly the editing efficiency of the system.

中文翻译：

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使用 CRISPR-Cpf1 在 Ashbya gossypii 中进行多重基因组编辑

CRISPR/Cas 技术构成了包括真菌在内的许多生物体的快速基因组工程的重要工具。适用于工业真菌 Ashbya gossypii 的 CRISPR/Cas9 系统可实现高效的基因组编辑，以引入缺失、插入和核苷酸替换。然而，Cas9 系统受到目标站点中特定 5'-NGG-3' PAM 序列的限制。在这里，我们展示了一种新的 CRISPR/Cas 系统，用于扩展可用于这种真菌微生物工程的分子工具箱。使用来自毛螺科细菌的 Cpf1 核酸酶允许采用富含 T 的 PAM 序列 (5'-TTTN-3')，并促进适用于棉线菌的多路 CRISPR/Cpf1 系统的实施。该系统已通过五种不同营养缺陷标记（HIS3、ADE2、TRP1、LEU2 和 URA3）引入大缺失的验证。在多 CRISPR/Cpf1 系统中同时使用 crRNA 和 dDNA 阵列被证明是使用单个多 CRISPR/Cpf1 质粒对多达四个基因进行多重基因缺失的有效策略。我们的结果还表明，目标序列的选择可能会显着影响系统的编辑效率。