ANDAMIOS Y PLATAFORMAS ELEVADAS

Several approaches have been utilised to induce targeted gene silencing and gene knockout in mammalian cells. Since shRNA was first manipulated to induce targeted gene silencing (Fire et al. 1998) it has been widely used to silence the expression of many genes. More recently, advances within the nuclease gene editing field have produced gene editing tools that induce gene knockout through the DSBs which when repaired by erroneous pathways lead to the introduction of mutations at a DNA level. Chronologically, ZFNs were identified first, followed by TALENs and most recently the CRISPR/Cas9 system. Each of these gene editing tools has been used widely since their discovery and each new technology has largely superseded its predecessors as the methodology of choice. As part of this project, plasmid systems were developed for each gene editing tool targeting the CD8A gene in order to validate and compare each of the systems. The TALEN approach (section 3.1.4) was not included in this study; the limitations of TALENs are discussed in section 3.4.2. The shRNA, ZFN and CRISPR/Cas9 approaches were validated and compared. Each of the approaches successfully induced gene silencing or gene knockout in the Molt3 cell line. The shRNA approach did not result in consistent gene silencing as the frequency of CD8 negative cells rose substantially throughout the monitoring period. The ZFN approach led to an approximate knockout efficiency of 20%, which was fairly consistent across the monitoring period. The CRISPR/Cas9 approach was found to lead to superior efficiency of gene knockout with approximately 80% of transduced cells expressing CD8A at levels equivalent to the FMO control consistently across the monitoring period when monitored by flow cytometry.

3.4.1 Use of double reporter system for identification of cells transduced

with both the left and right ZFN constructs

In this thesis the ZFNs delivered by lentiviral transduction resulted in the knockout of CD8 from the Molt3 cell line as demonstrated by flow cytometry. However, the frequency of cells expressing both the LZFN and RZFN proteins was not established as a GFP marker was present in both constructs and therefore single and double transductants were indistinguishable. To improve this system for future use a second reporter gene could be

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cloned into one of the ZFN constructs to allow for detection of cells transduced with both the LZFN and RZFN.

3.4.2 Generation of TALENs

Assembly of TALENs was attempted using two commercially available kits by Cermak et al. 2011 and Sanjana et al. 2012 (Methods can be found in Appendix Figure 2 and 3. The process of TALEN construction can be found in Appendix Figure 4 and 5). A TALEN targeting the CD8A gene was produced by the Cermak Kit (Appendix Figure 4); however, no TALENs were produced with the Sanjana kit (Appendix Figure 5). The TALEN approach was incompatible with lentiviral transduction due to unintentional rearrangements within the TALE sequence (Holkers et al. 2012). A pair of TALENs were synthesised (Eurofins) which were designed to minimise the repetitive nature of the TALE arrays. The TALE protein was cloned into the pRRL.sin.cppt.pgk-gfp.wpre lentivector backbone which contained a GFP reporter. However, attempts to transduce cells with the TALE protein were unsuccessful as no GFP was expressed (Appendix Figure 6). I spent considerable time in design and testing of TALENS. Once CRISPR came along I switched my efforts to this new tool.

3.4.3 Design and construction of gene editing tools

In order to generate shRNA targeting the CD8A gene for this project the target sequence published by the Broad institute was synthesised (Eurofins) and cloned into a lentiviral backbone. This process is fast and inexpensive as shRNA constructs can be generated within 2 weeks dependent upon the synthesis of the target sequence. The ZFNs used in this project were produced by Sigma Aldrich, unless ZFNs targeting the gene of interest have previously been ordered from the company the production costs and timing are increased as due to the bespoke validation process. ZFNs for novel gene targets can take several weeks to produce and cost several thousand pounds per pair. Once the ZFN plasmid is delivered the gene must be cloned into a lentiviral backbone. The newer, CRISPR/Cas9 technology is by far the easiest and cheapest approach as altering the specificity of the simply involves design and ordering crRNA sequences. The oligos typically cost less than ten pounds per pair. The oligos are then cloned into the CRISPR/Cas9 lentiviral backbone, from start to finish this process can be completed within a week. Unless validated crRNA sequences for target genes

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are published there is no guarantee that the crRNA sequences produced will be functional, however, using this system within our lab we have produced crRNAs targeting a range of genes and have found that designing and producing 5 crRNAs per target gene results in a minimum of 2 functional crRNAs. The efficiency, ease and low cost of CRISPR/Cas9 has resulted in this system now being the method of choice for gene silencing and the approach was awarded Breakthrough of the Year 2015 by Science magazine (Travis 2015).

3.4.4 Gene silencing efficiency

When directly compared against one another there was a reduction of MFI of 98, 100 and 93% for shRNA, ZFN and CRISPR/Cas9 respectively. Although the reduction in MFI was comparable between these gene editing tools the frequency of CD8 negative cells in the transduced populations varied. At two weeks post transduction, the CRISPR/Cas9 approach resulted in a knockout efficiency of 84% in transduced cells. The ZFN approach had an efficiency of 20% while 67% of cells transduced with the shRNA approach had observable CD8 gene silencing. The level of CD8 expressed by the cells transduced with the shRNA did not remain silenced and at five weeks post transduction, only 35% of the transduced cells expressed CD8 at low levels.

3.4.5 Summary

In this chapter I began by comparing shRNA, TALEN and ZFN technologies for silencing of the CD8A gene. I put considerable effort into building the TALEN tools, unfortunately, these efforts were unfruitful. While struggling with TALENs, CRISPR/Cas9 technology became available so I adopted to utilise this methodology. Overall, in direct comparisons, the CRISPR technology proved to be preferable. I went on to use CRISPR technology to study antigen presentation to unconventional T-cells in chapter 6. Some concerns have been raised about the use of nuclease based gene editing for clinical application. I next set out to find an alternative technology that did not incorporate the inherent risks of off-target nuclease activity. I will further discuss the implications of all this work in chapter 4.

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