Programming cells by multiplex genome engineering and.pdfVIP

doi:10.1038/nature08187 LETTERS Programming cells by multiplex genome engineering and accelerated evolution 1,2,3 1 4,5 6 6 7 Harris H. Wang *, Farren J. Isaacs *, Peter A. Carr , Zachary Z. Sun , George Xu , Craig R. Forest George M. Church1 The breadth of genomic diversity found among organisms in nature of the replication fork during DNA replication14. We optimized a allows populations to adapt to diverse environments1,2. However, number of parameters (see Supplementary Information, Supplemen- genomic diversity is difficult to generate in the laboratory and new tary Fig. 2 and Supplementary Table 1) to maximize efficiency of phenotypes do not easily arise on practical timescales3. Although in oligo-mediated allelic replacement. To generate sequence diversity vitro and directed evolution methods4–9 have created genetic in any region of the chromosome by allelic replacement, a pool of variants with usefully altered phenotypes, these methods are limited targeting oligos is repeatedly introduced into a cell. Under optimized to laborious and serial manipulation of single genes and are not used conditions, we can successfully introduce new genetic modifications for parallel and continuous directed evolution of gene networks or in .30% of the cell population (Supplementary Fig. 2d) every genomes. Here, we describe multiplex automated genome engineer- 2–2.5 h. ing (MAGE) for large-scale programming and evolution of cells. Oligo-mediated allelic replacement is capable of intr