Scalable and automated CRISPR-based strain engineering using droplet microfluidics  

作　　者：Kosuke Iwai Maren Wehrs Megan Garber Jess Sustarich Lauren Washburn Zachary Costello Peter W.Kim David Ando William R.Gaillard Nathan J.Hillson Paul D.Adams Aindrila Mukhopadhyay Hector Garcia Martin Anup K.Singh 

机构地区：[1]Technology Division,DOE Joint BioEnergy Institute,Emeryville,CA 94608,USA [2]Biotechnology and Bioengineering Department,Sandia National Laboratories,Livermore,CA 94550,USA [3]Biofuels and Bioproducts Division,DOE Joint BioEnergy Institute,Emeryville,CA 94608,USA [4]Biological Systems and Engineering Division,Lawrence Berkeley National Laboratory,Berkeley,CA 94720,USA [5]Molecular Biophysics and Integrated Bioimaging Division,Lawrence Berkeley National Laboratory,Berkeley,CA 94720,USA [6]Department of Bioengineering,University of California,Berkeley,CA 94720,USA [7]BCAM,Basque Center for Applied Mathematics,Bilbao 48009,Spain

出　　处：《Microsystems & Nanoengineering》2022年第2期227-236,共10页微系统与纳米工程（英文）

基　　金：supported by the Basque Government through the BERC 2014-2017 program and the Spanish Ministry of Economy and Competitiveness MINECO through the BCAM Severo Ochoa excellence accreditation SEV-2013-0323;Sandia National Laboratories is a multimission laboratory managed and operated by National Technology and Engineering Solutions of Sandia,LLC,a wholly owned subsidiary of Honeywell International,Inc.,for the U.S.Department of Energy's National Nuclear Security Administration under contract DE-NA-0003525.

摘　　要：We present a droplet-based microfluidic system that enables CRISPR-based gene editing and high-throughput screening on a chip.The microfluidic device contains a 10x10 element array,and each element contains sets of electrodes for two electric field-actuated operations:electrowetting for merging droplets to mix reagents and electroporation for transformation.This device can perform up to 100 genetic modification reactions in parallel,providing a scalable platform for generating the large number of engineered strains required for the combinatorial optimization of genetic pathways and predictable bioengineering.We demonstrate the system's capabilities through the CRISPR-based engineering of two test cases:(1)disruption of the function of the enzyme galactokinase(galK)in£coli and(2)targeted engineering of the glutamine synthetase gene(glnA)and the blue-pigment synthetase gene(bpsA)to improve indigoidine production in£coli.

关 键 词：operations GENERATING FLUID 

分 类 号：O35[理学—流体力学]