Developing Methods to Circumvent the Conundrum of Chromosomal Rearrangements Occurring in Multiplex Gene Edition.,ACS Synthetic Biology

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Developing Methods to Circumvent the Conundrum of Chromosomal Rearrangements Occurring in Multiplex Gene Edition.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-08-07 , DOI: 10.1021/acssynbio.0c00325
Vinciane Borsenberger ₁ , Christian Croux ₁ , Fayza Daboussi _{1,

2} , Cécile Neuvéglise ₃ , Florence Bordes ₁

Affiliation

CRISPR/Cas9 is a powerful tool to edit the genome of the yeast Yarrowia lipolytica. Here, we design a simple and robust method to knockout multiple gene families based on the construction of plasmids enabling the simultaneous expression of several sgRNAs. We exemplify the potency of this approach by targeting the well-characterized acyl-CoA oxidase family (POX) and the uncharacterized SPS19 family. We establish a correlation between the high lethality observed upon editing multiple loci and chromosomal translocations resulting from the simultaneous generation of several double-strand breaks (DSBs) and develop multiplex gene editing strategies. Using homologous directed recombination to reduce chromosomal translocations, we demonstrated that simultaneous editing of four genes can be achieved and constructed a strain carrying a sextuple deletion of POX genes. We explore an “excision approach” by simultaneously performing two DSBs in genes and reached 73 to 100% editing efficiency in double disruptions and 41.7% in a triple disruption. This work led to identifying SPS193 as a gene encoding a 2–4 dienoyl-CoA reductase, demonstrating the potential of this method to accelerate knowledge on gene function in expanded gene families.

中文翻译：

开发方法来规避多重基因版中发生的染色体重排难题。

CRISPR/Cas9 是编辑解脂耶氏酵母基因组的强大工具。在这里，我们设计了一种简单而稳健的方法，基于质粒的构建来敲除多个基因家族，从而能够同时表达多个 sgRNA。我们通过针对已充分表征的酰基辅酶 A 氧化酶家族 (POX) 和未表征的 SPS19 家族来举例说明该方法的效力。我们建立了编辑多个位点时观察到的高致死率与同时产生多个双链断裂（DSB）导致的染色体易位之间的相关性，并开发了多重基因编辑策略。利用同源定向重组减少染色体易位，我们证明可以实现四个基因的同时编辑，并构建了携带 POX 基因六重缺失的菌株。我们探索了一种“切除方法”，通过在基因中同时执行两个 DSB，在双重破坏中达到 73% 至 100% 的编辑效率，在三重破坏中达到 41.7% 的编辑效率。这项工作确定了 SPS193 是编码 2-4 二烯酰辅酶 A 还原酶的基因，证明了该方法在加速了解扩展基因家族中基因功能的潜力。

更新日期：2020-09-20

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