Chemical and Biomolecular Engineering, Clemson University, Clemson, South Carolina, USA.
Biotechnol J. 2018 Sep;13(9):e1700590. doi: 10.1002/biot.201700590. Epub 2018 Jun 11.
CRISPR-Cas9 technology has been successfully applied in Yarrowia lipolytica for targeted genomic editing including gene disruption and integration; however, disruptions by existing methods typically result from small frameshift mutations caused by indels within the coding region, which usually resulted in unnatural protein. In this study, a dual cleavage strategy directed by paired sgRNAs is developed for gene knockout. This method allows fast and robust gene excision, demonstrated on six genes of interest. The targeted regions for excision vary in length from 0.3 kb up to 3.5 kb and contain both non-coding and coding regions. The majority of the gene excisions are repaired by perfect nonhomologous end-joining without indel. Based on this dual cleavage system, two targeted markerless integration methods are developed by providing repair templates. While both strategies are effective, homology mediated end joining (HMEJ) based method are twice as efficient as homology recombination (HR) based method. In both cases, dual cleavage leads to similar or improved gene integration efficiencies compared to gene excision without integration. This dual cleavage strategy will be useful for not only generating more predictable and robust gene knockout, but also for efficient targeted markerless integration, and simultaneous knockout and integration in Y. lipolytica.
CRISPR-Cas9 技术已成功应用于解脂耶氏酵母（Yarrowia lipolytica）的靶向基因组编辑，包括基因敲除和整合；然而，现有方法的敲除通常是由于编码区的插入缺失导致的小移码突变，这通常会导致非天然蛋白质。在本研究中，开发了一种由成对 sgRNA 指导的双切割策略用于基因敲除。该方法允许快速、稳健的基因切除，在六个感兴趣的基因上得到了验证。切除的靶区域长度从 0.3kb 到 3.5kb 不等，包含非编码区和编码区。大多数基因的切除都是通过无插入缺失的完美非同源末端连接修复的。基于该双切割系统，通过提供修复模板开发了两种靶向无标记整合方法。虽然这两种策略都有效，但基于同源介导末端连接（HMEJ）的方法比基于同源重组（HR）的方法效率高两倍。在这两种情况下，与不进行整合的基因切除相比，双切割导致类似或提高的基因整合效率。这种双切割策略不仅可用于产生更可预测和稳健的基因敲除，还可用于高效的靶向无标记整合，以及在解脂耶氏酵母中同时进行基因敲除和整合。
