Smith Justin D, Schlecht Ulrich, Xu Weihong, Suresh Sundari, Horecka Joe, Proctor Michael J, Aiyar Raeka S, Bennett Richard A O, Chu Angela, Li Yong Fuga, Roy Kevin, Davis Ronald W, Steinmetz Lars M, Hyman Richard W, Levy Sasha F, St Onge Robert P
Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.
Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.
Mol Syst Biol. 2017 Feb 13;13(2):913. doi: 10.15252/msb.20167233.
The low costs of array-synthesized oligonucleotide libraries are empowering rapid advances in quantitative and synthetic biology. However, high synthesis error rates, uneven representation, and lack of access to individual oligonucleotides limit the true potential of these libraries. We have developed a cost-effective method called Recombinase Directed Indexing (REDI), which involves integration of a complex library into yeast, site-specific recombination to index library DNA, and next-generation sequencing to identify desired clones. We used REDI to generate a library of ~3,300 DNA probes that exhibited > 96% purity and remarkable uniformity (> 95% of probes within twofold of the median abundance). Additionally, we created a collection of ~9,000 individually accessible CRISPR interference yeast strains for > 99% of genes required for either fermentative or respiratory growth, demonstrating the utility of REDI for rapid and cost-effective creation of strain collections from oligonucleotide pools. Our approach is adaptable to any complex DNA library, and fundamentally changes how these libraries can be parsed, maintained, propagated, and characterized.
阵列合成寡核苷酸文库的低成本推动了定量生物学和合成生物学的快速发展。然而，高合成错误率、代表性不均以及无法获取单个寡核苷酸限制了这些文库的真正潜力。我们开发了一种具有成本效益的方法，称为重组酶定向索引（REDI），该方法包括将复杂文库整合到酵母中、进行位点特异性重组以索引文库DNA以及使用下一代测序来鉴定所需克隆。我们使用REDI生成了一个约3300个DNA探针的文库，该文库纯度大于96%，且具有显著的均匀性（超过95%的探针丰度在中位数的两倍范围内）。此外，我们为发酵或呼吸生长所需的超过99%的基因创建了一个约9000个可单独获取的CRISPR干扰酵母菌株的集合，证明了REDI在从寡核苷酸库快速且经济高效地创建菌株集合方面的实用性。我们的方法适用于任何复杂的DNA文库，并从根本上改变了这些文库的解析、维护、繁殖和表征方式。
