RNA-guided gene drives capable of spreading genomic alterations made in laboratory organisms through wild populations in an inheritable way could be used to control populations of organisms that cause environmental and public health problems. However, the possibility of unintended genome editing through the escape of strains from laboratories, coupled with the prospect of unanticipated ecological change, demands caution. We report the efficacy of CRISPR-Cas9 gene drive systems in wild and laboratory strains of the yeast Saccharomyces cerevisiae. Furthermore, we address concerns surrounding accidental genome editing by developing and validating methods of molecular confinement that minimize the risk of unwanted genome editing. We also present a drive system capable of overwriting the changes introduced by an earlier gene drive. These molecular safeguards should enable the development of safe CRISPR gene drives for diverse organisms.Synthetic gene drive systems have the potential to address diverse ecological problems by altering the traits of wild populations. These genetic elements spread not by improving the reproductive fitness of the organism, but by increasing the odds that they themselves will be inherited. Because this inheritance advantage can overcome the fitness costs associated with the drive itself or adjacent genes carried with the drive, such genetic elements are theoretically capable of 'driving' unrelated traits through populations over many generations 1 .Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptInheritance-biasing is a common strategy in nature 2 . One elegant class of inheritance-biasing genes spreads by cutting homologous chromosomes that do not contain them, thereby inducing the cellular repair process to copy them onto the damaged chromosome by homologous recombination (Fig. 1A). This process is known as 'homing' 3 . The bestcharacterised homing endonuclease gene is I-SceI, whose product cuts the gene encoding the large rRNA subunit of S. cerevisiae mitochondria. Most homing endonucleases are extremely efficient, for example, I-SceI is correctly copied 99% of the time 4 .Austin Burt suggested in 2003 that homing endonucleases might form the basis of synthetic gene drives that could alter wild populations of sexually reproducing organisms (Fig. 1B) 5 .The I-SceI endonuclease gene was subsequently demonstrated to exhibit homing in transgenic laboratory populations of mosquitoes 6 and fruit flies 7 , 8 in which an I-SceI recognition site was inserted into the recipient strain at the same locus as the I-SceI gene in the donor strain.. However, the difficulty of retargeting homing endonucleases to cleave specific sequences in wild-type genomes has limited their utility for synthetic gene drive elements 9 .CRISPR-Cas9 which cleaves target sequences specified by single 'guide RNA' (sgRNA) mo...

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Inheritance-biasing elements known as "gene drives" may be capable of spreading genomic alterations made in laboratory organisms through wild populations. We previously considered the potential for RNA-guided gene drives based on the versatile CRISPR/Cas9 genome editing system to serve as a general method of altering populations 1 . Here we report molecularly contained gene drive constructs in the yeast Saccharomyces cerevisiae that are typically copied at rates above 99% when mated to wild yeast. We successfully targeted both non-essential and essential genes and showed that the inheritance of an unrelated "cargo" gene could be biased by an adjacent drive. Our results demonstrate that RNA-guided gene drives are capable of efficiently biasing inheritance when mated to wild-type organisms over successive generations.

The first promising results from "streamlined," minimal genomes tend to support the notion that these are a useful tool in biological systems engineering. However, compared with the speed with which genomic microbial sequencing has provided us with a wealth of data to study biological functions, it is a slow process. So far only a few projects have emerged whose synthetic ambition even remotely matches our analytic capabilities. Here, we survey current technologies converging into a future ability to engineer large-scale biological systems. We argue that the underlying synthetic technology, de novo DNA synthesis, is already rather mature - in particular relative to the scope of our current synthetic ambitions. Furthermore, technologies towards rationalizing the design of the newly synthesized DNA fragment are emerging. These include techniques to implement complex regulatory circuits, suites of parts on a DNA and RNA level to fine tune gene expression, and supporting computational tools. As such DNA fragments will, in most cases, be destined for operating in a cellular context, attention has to be paid to the potential interactions of the host with the functions encoded on the engineered DNA fragment. Here, the need of biological systems engineering to deal with a robust and predictable bacterial host coincides with current scientific efforts to theoretically and experimentally explore minimal bacterial genomes.

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