Zhang Qiaoxin, Ren Junle, Wu Siyao, Tan Yadi, Wang Wenliang, Feng Cai, Zhao Liqian, Zhu Zhiwei
MOE Key Laboratory of Bio-Intelligent Manufacturing, School of Bioengineering, Dalian University of Technology, Dalian, Liaoning, China.
School of Environmental Science and Technology, Dalian University of Technology, Dalian, Liaoning, China.
Biotechnol Bioeng. 2025 Sep 9. doi: 10.1002/bit.70062.
Plasmids are commonly employed in the delivery of clustered regularly interspaced shortpalindromic repeats (CRISPR)/CRISPR-associated (Cas) components for genome editing. However, the absence of heritable plasmids in numerous organisms limits the development of CRISPR/Cas genome editing tools. Moreover, cumbersome procedures for plasmid construction and curing render genome editing time-consuming. In this study, we developed a plasmid-free CRISPR/Cpf1 genome editing system for Saccharomyces cerevisiae and Starmerella bombicola. This system leveraged integrative expression of the Cpf1 nuclease and T7 RNA polymerase (T7RNAP), as well as the delivery of linear fragments including (i) a marker cassette for integration and selection, (ii) short double-stranded DNA (crDNA) for in vivo transcription of crRNA by T7RNAP, and (iii) donor DNA for homology-directed repair. We demonstrated that this editing system enabled efficient multiplexed and iterative genome editing without the need for marker recycling and plasmid curing. The use of short crDNA (87 bp) and donor DNA (≤ 120 bp), both readily prepared from ordered oligonucleotides via annealing or overlap extension, dramatically simplified the editing process. Successful implementation in S. bombicola, which lacks heritable plasmids for genetic engineering, highlighted the potential of this approach especially for genome engineering of genetically intractable organisms in a plasmid-free way.
质粒常用于递送成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas）组件以进行基因组编辑。然而，许多生物体中缺乏可遗传的质粒限制了CRISPR/Cas基因组编辑工具的发展。此外，繁琐的质粒构建和消除程序使得基因组编辑耗时。在本研究中，我们为酿酒酵母和博伊丁假丝酵母开发了一种无质粒的CRISPR/Cpf1基因组编辑系统。该系统利用了Cpf1核酸酶和T7 RNA聚合酶（T7RNAP）的整合表达，以及线性片段的递送，这些线性片段包括：（i）用于整合和筛选的标记盒，（ii）用于T7RNAP在体内转录crRNA的短双链DNA（crDNA），以及（iii）用于同源定向修复的供体DNA。我们证明，该编辑系统能够实现高效的多重和迭代基因组编辑，而无需标记回收和质粒消除。使用短crDNA（87 bp）和供体DNA（≤120 bp），二者均可通过退火或重叠延伸从定制的寡核苷酸轻松制备，极大地简化了编辑过程。在缺乏用于基因工程的可遗传质粒的博伊丁假丝酵母中的成功应用，突出了这种方法的潜力，特别是对于以无质粒方式对遗传操作困难的生物体进行基因组工程的潜力。
