MUSERGEN: MultiUSER equipment for GENe identification in biosciences and biotechnology

Improving our understanding of biology is largely accomplished by natural or laboratory manipulation of the sequences of genes (and hence proteins), and observations of the effects on the organisms of interest. Such effects can include tolerance to various stresses, the ability to metabolise compounds of interest, the ability to improve longevity, to remain healthy, and so on. In principle one might do this many times to produce vast libraries of variants. If the organism is one that makes a product of interest (e.g. penicillin, a biofuel, or indeed anything else), this also leads to the possibility of improving the host's productivity dramatically.

In practice, most methods are able only to remove (knock out) the activity of genes, but fewer can be used to manipulate them in a graded manner, and this tends to be done in a bespoke or 'craft' industry kind of way.

More recently, it was discovered that a system of 'immune defence' for bacteria against certain viruses, known as CRISPR-Cas9, allows a much more precise 'gene editing', and this general strategy is revolutionising biology. Again, however, the edits tend to be done one at a time.

Very recently (October 1st, 2019), an instrument became available that is able to perform such gene editing on a genome-wide scale, allowing thousands of such experiments effectively to be done at once (in parallel). It uses a version of CRISPR-Cas9 with its own proprietary system (but one that is entirely free for commercial use). By tagging each known edit with a barcode, and assessing the frequency with which particular barcodes are expressed under a condition of interest, one can assess the relative importance of hundreds of genes simultaneously, and how they should be modified. This instrument is designed to 'democratise' CRISPR-based gene editing, and it is known as the Inscripta Onyx instrument. The purpose of this proposal is to obtain funds to purchase such an instrument, to set it up in Liverpool's synthetic biology centre (the Gene Mill in the Centre for Genomic Research), and to make it available to the local and wider BBSRC community.

The result of this program will be huge amounts of new knowledge of which genes - which will often be unexpected ones - are involved in various bioprocesses of interest. It will allow us to control and optimize cellular enzyme and small molecule production in a truly rational manner.

Biology mainly advances by relating the sequences of genes (and hence proteins) to their phenotypic effects. If the organism is one that makes a product of interest (e.g. penicillin, a biofuel, or indeed anything else), this also leads to the possibility of improving the host's productivity dramatically.

In practice, most methods are able only to remove (knock out) the activity of genes, but far fewer can be used to manipulate them in a graded manner. This also tends to be done serially in a bespoke or 'craft' kind of way.

CRISPR-Cas9 and related technologies allow a much more precise 'gene editing', and this general strategy is revolutionising biology. Again, however, the edits tend to be done one at a time.

Very recently (October 1st, 2019), an instrument became available that is able to perform such gene editing on a genome-wide scale, allowing thousands of such experiments effectively to be done at once (in parallel). It uses a version of CRISPR-Cas9 with its own proprietary system (but one that is entirely free for commercial use). By tagging each known edit with a barcode, and assessing the frequency with which particular barcodes are expressed under a condition of interest, one can assess the relative importance of hundreds of genes simultaneously, and how they should be modified. This Inscripta Onyx instrument is designed to 'democratise' CRISPR-based gene editing, and is fully automated. The purpose of this proposal is to obtain funds to purchase such an instrument, to set it up in Liverpool's synthetic biology centre (the Gene Mill in the Centre for Genomic Research), and to make it available to the local and wider BBSRC community.

The result of this program will be huge amounts of new knowledge of which genes - which will often be unexpected ones - are involved in a bioprocess of interest. It will allow us to control and optimise cellular enzyme and small molecule production in a truly rational manner.

WHO WILL BENEFIT: The scientific community will benefit in a number of ways, by gaining access to a genuinely breakthrough technology that allows scientists to enable the immense changes that can be effected via the use of synbio methods on a genome-wide scale, along with the recognition that the differential influence (not just all up or all down) is what underlies this. Evidence that this really is a breakthrough technology comes from the fact that Inscripta has already raised over $130M (evidence at https://www.crunchbase.com/organization/inscripta#section-overview).

So far as biotechnology more generally is concerned, companies will benefit from knowledge of the benefits of these approaches for genome-wide refactoring and its utility in improving the production of desirable enzymes and small molecules. Quantitative models of biochemical networks networks are still in their infancy, and efforts in this direction are largely data-limited. By providing high-throughput, highly quantitative datasets to the community, we will advance the field and allow others to build on this work.

HOW WILL THEY BENEFIT: As is our practice, all pertinent data are made available in public databases, and OA publishing has long been our norm. We shall also hold frequent workshops in Liverpool and elsewhere, to assist dissemination of this technology and our ownresearch results. We have pioneered in the Altmetrics field for digital dissemination: indeed, in a recent Nature article (Altmetrics make their mark. Nature 2013; 500:491-492) Kwok highlighted the fact that the PI's paper Hull D, Pettifer SR, Kell DB: Defrosting the digital library: bibliographic tools for the next generation web. PLoS Comput Biol 2008; 4:e1000204, was the most accessed ever in any PLoS journal, with over 53,000 accesses (it is past 110,000 now). Another (Open Access paper) has just passed 100,000 (Kell DB: Iron behaving badly: inappropriate iron chelation as a major contributor to the aetiology of vascular and other progressive inflammatory and degenerative diseases. BMC Med Genom 2009, 2:2). We shall work closely with University KT staff and industrial IP offices as part of this project. Finally, having secured any IP, we shall, of course, seek actively to communicate our scientific findings to the wider research community through scientific meetings, scholarly publications and press releases.

THE WIDER COMMUNITY: DBK (@dbkell) and many of the other applicants are well known bloggers and tweeters, and social media will provide a novel and useful means of disseminating our findings.

COMMUNICATIONS: We have already made a video describing our recent novel method for AMR https://umip.com/current-technologies/our-technologies/novel-method-for-rapid-antimicrobial-susceptibility-testing/, and we have commissioned SciAni Ltd to produce an animation of our membrane transporter projects. We will communicate with relevant industrial and academic partners both directly and via the meetings of relevant learned societies (we are members of several). 2020 is the 75th anniversary of the Microbiology Society, and (as a previous Fleming lecturer) the PI has already been invited to attend this major meeting. In year three of the Project, we will organise a half-day meeting to explain our research to interested industrial scientists. However, we will also provide a video link to facilitate the participation of those who are unable to travel to Liverpool.