Genomics CRISPR/Cas9 – transforming gene editing in drug discovery labs It is evident from a recent market survey on gene editing in drug discovery that CRISPR/Cas9 is now recognised as the superlative method when attempting a gene knockout or when introducing defined mutations, insertions or modifications to the genome. Although use of CRISPR/Cas9 is still mainly confined to the basic research area in the drug discovery process, its potential with respect to the identification and validation of new therapeutic targets, the investigation of mechanism of action and in the creation of screens to identify genes that regulate various cell biological processes are major objectives. The perceived key advantages of CRISPR/Cas9 edits are their efficiency (ie it edits targets sequences at surprisingly high rates), simplicity (ie easy to use and design) and programmability (ie get precision targeting). The main benefits of CRISPR/Cas9 technology that drug discovery labs are most eager to exploit is the ability to make a complete genetic knockout, while minimising off-target effects; the rapid generation of cell lines harbouring desired mutations; and the possibility to develop accurate models of complex human disease in an efficient manner. The delivery of the CRISPR components into some target cells remains sub-optimal and this limits some work on CRISPR/Cas9 gene editing today. Overall, there seems little doubt that CRISPR/Cas9 technology will transform gene editing in drug discovery labs over the coming years. enome editing uses engineered nucleases The CRISPR/Cas 9 system takes advantage of a By Dr John Comley in conjunction with endogenous repair s hort guide RNA (gRNA) to target the bacterial G mechanisms to alter the DNA in a cell. Cas9 endonuclease to specific genomic loci. Recently, a new genome editing tool based on a Because the gRNA supplies the specificity, chang- bacterial-clustered regularly-interspaced short ing the target only requires a change in the design palindromic repeats (CRISPR) and its associated of the sequence encoding the gRNA, as a result protein-9 nuclease (Cas9) has generated consider- editing can be directed to virtually any genomic able interest and looks set to transform the field. locus including most eukaryotic cells. The Drug Discovery World Fall 2016 33 Genomics CRISPR/Cas9 system greatly simplifies genome Figure 1: Current experience of gene editing editing and has great promise in many applications with CRISPR/Cas 9 technology areas such as stem cell engineering, gene therapy, Cutting edge – expert at drug development, tissue and animal disease mod- the forefront of current research els, agriculture, plant disease resistance, etc. In 4% drug development CRISPR can provide a more High – using regularly Very limited – complete 5% effective method to probe the function of specific novice genes, such that drug discovery and validation can 32% Moderate – some practical experience be accelerated. In gene therapy CRISPR offers pre- 22% cise editing or knockout of specific segments of a genome, enabling genetic research of defective genes and their behaviour. In February 2016, HTStec undertook a market Low – have made some initial investigations survey on CRISPR/Cas9 gene editing in drug dis- 37% covery mainly among drug research labs in phar- © HTStec 2016 ma, biotech and academia1. The main objective of this survey was to document the current use of CRISPR/Cas9 in gene editing in drug discovery applications and to understand its future impact. In Figure 2: Time period since first started using this article highlights from the market survey are CRISPR/Cas9 gene editing technology reported and the findings are discussed together with vendor updates on their CRISPR/Cas9 offer- 40% ings supporting drug discovery research. 38% 35% Current use of CRISPR/ 30% Cas9 gene editing 25% Most survey respondents’ current experience of 20% gene editing with CRISPR/Cas 9 technology and its % Responding 15% potential in drug discovery was low, ie have made 16% 15% some initial investigations. This was followed by 10% 9% 8% 32% very limited, ie complete novice; 22% moder- 5% 7% 7% ate, ie some practical experience; 5% high, ie using 0% N/A – not yet using <3 months 3-6 months 6-12 months 12-18 months 18-24 months >2 years regularly; and then only 4% cutting edge, ie expert at the forefront of current research (Figure 1). The © HTStec 2016 median time period since survey respondents had first investigated or started using CRISPR/Cas 9 gene editing technology was 6-12 months ago (Figure 2). Most (47%) of survey respondents were Figure 3: Area of the drug discovery process applying or intending to apply CRISPR/Cas 9 gene most applying or intending to apply editing to the basic research area of the drug dis- covery process. This was followed by target valida- CRISPR/Cas 9 gene editing tion (18% applying); target identification (10% applying) and then clinical studies and lead gener- Basic research 47% ation (both with 9% applying). Other drug discov- Target validation 18% ery areas ha d in total only 6% applying (Figure 3). Target identification 10% Oncology/cancer was the key disease or research Clinical studies 9% area most targeted by survey respondents (52% Lead generation 9% targeting) by CRISPR/Cas 9 gene editing. This was Biologics and bioprocessing 3% followed by immunology/inflammatory disease/ ADME/toxicology 2% autoimmune (30% targeting); neurology/CNS/ Lead optimisation 1% neurodegeneration/pain (27% targeting); metabol- 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% ic disease/diabetes (18% targeting); and then car- % Responding diovascular disease (17% targeting) (Figure 4). Most (49%) of survey respondents answered N/A, © HTStec 2016 ie they have not investigated other gene editing 34 Drug Discovery World Fall 2016 Genomics technologies prior to CRISPR/Cas 9 availability. 29% have previously investigated transcription Figure 4: Key diseases or research areas using activator-like effector nucleases (TALENs); 21% CRISPR/Cas 9 gene editing have investigated integration via homologous recombination (eg with rAAV); 16% zinc finger Oncology/cancer 50% Immunology/inflammatory nucleases (ZFN); and 11% other approaches disease/autoimmune 29% (Figure 5). Neurology/CNS/ 26% neurodegeneration/pain Metabolic disease/diabetes 18% Main rationale for using CRISPR/ Cas9 gene editing technology Cardiovascular disease 17% A gene knockout was what the majority (77%) of Systems biology 14% survey respondents most want to achieve using Respiratory/pulmonary disease 11% CRISPR/Cas 9 gene editing technology. This was Other 10% followed by introduce defined mutations, inser- Infectious diseases 10% tions or modifications (62% wanting); gene Bone and skeletal disease 8% knock-in (52% wanting); and then gene knock Toxicology 7% down (inducible) (40% wanting).Other aims were wanted by less than a third of survey respondents Drug metabolism studies 6% Not applying to any specific (Figure 6). Identification of new therapeutic targets disease or research area 5% was the main objective of survey respondents 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% 55% CRISPR/Cas 9 gene editing in drug discovery % Responding © HTStec 2016 (61% wanting). This was followed by validation of new therapeutic targets (48% wanting); and then investigation of mechanism of action and screens Figure 5: Other gene editing technologies to identify genes that regulate various cell biologi- investigated prior to CRISPR/Cas 9 cal processes (both with 46% wanting). Of least interest was deconvolution and validation of N/A – did not attempt gene GWAS hits (only 8% targeting) (Figure 7). Survey editing prior to CRISPR/Cas9 47% respondents ranked some of the perceived advan- Transcription activator-like effector nucleases (TALENs) 28% tages of CRISPR/Cas 9 gene editing technology in Integration via homologous 20% order of importance. This analysis revealed that recombination (eg with rAAV) efficient, ie edit targets sequences at surprisingly Zinc finger nucleases (ZFN) 16% high rates, was seen as the main advantage of Other approaches 11% CRISPR/Cas 9 gene editing technology. This was followed by simplicity, ie easy to use and design; 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% % Responding programmable, ie get precision targeting; and then © HTStec 2016 fast, ie get tangible results wi thin weeks. Ranked least advantageous was multiplexing, ie can pro- gramme multiple guide RNAs and cleave multiple Figure 6: What respondents want to achieve genes simultaneously (Figure 8). Survey respon- using CRISPR/Cas 9 gene editing dents rated complete genetic knockout, while min- imising off-target effects as the potential benefit of CRISPR/Cas 9 in drug discovery they were most Gene knockout 77% interested in exploiting. This was closely followed Introduce defined mutations, insertions or modifications 62% by enables rapid generation of cell lines harbouring desired mutations; develop accurate models of Gene knock-in 52% complex human disease in an efficient manner; and Gene knock down (inducible) 40% then relative ease with which cellular models can Engineer induced pluripotent be generated. Rated least interesting was scalable stem cells (iPSCs) 33% generation of genome-wide CRISPR libraries for HT functional genomics screening (Figure 9). Introduce endogenous tags 30% Induce or repress transcription 25% Validating gene edits The downstream analytical technique most used to 0% 10% 20% 30% 40% 50% 60% 70% 80% © HTStec 2016 % Responding validate CRISPR/Cas 9 gene edits was P CR (70%