MATERIALES PELIGROSOS

This study sought to develop a cancer mouse model harbouring a 5.5 kb conditional KI within intron 11 of the Fbxw7 gene. The targeting construct used in this study contained a Cre-dependent conditional missense point mutation (R482Q) within exon 12 of Fbxw7. This point mutation is present in a diverse range of human cancers, including breast, lung and colorectal cancer (Babaei- Jadidi et al., 2011; Davis et al., 2011). A CRISPR/Cas9-meditated gene targeting strategy was used to generate mESCs for blastocyst microinjection. Direct gene targeting in mouse zygotes was not feasible due to the size and complexity of the Fbxw7 donor plasmid required to generate this large- scale conditional KI (17631 bp; Figure 2.2). Direct microinjection of donor plasmids into mouse zygotes have very low rates of integration, typically, 10 to 30% of zygotes carry the desired construct (Yang, Wang, & Jaenisch, 2014). As a CRISPR/Cas9-meditated gene targeting approach was employed, a gRNA targeting intron 11 in Fbxw7 was designed (Figure 4.1). On examination of the Fbxw7 target site it was evident that there were limited gRNA options that would not cleave of the Fbxw7 donor plasmid, whilst meeting the PAM requirements of SpCas9 and having an adequate predicted cutting efficiency. After systematic consideration, a gRNA was selected with a predicted cutting efficiency of 51 out of 100 at the Fbxw7 locus. This was calculated using the method developed by Fusi et al. (2015) and warranted further investigation to ensure sufficient cutting efficiency of the selected gRNA.

To experimentally investigate the cutting efficiency of the selected gRNA it was the tested in mouse melanoma cells. gRNA primers were subcloned into a CRISPR expression plasmid and the cloned plasmid was validated by restriction digestion before being confirmed by sequencing (Figure 4.2, 4.3 and 4.4). Melanocytes were then separately lipofected with the cCRISPR expression plasmid and a GFP expression plasmid. GFP expression in transfected cells confirmed an effective transfection protocol (Figure 4.5). Mouse melanoma cells successfully lipofected with the cCRISPR expression plasmid will express the unique gRNA and SpCas9, which, once complexed are capable of generating DSBs at the Fbxw7 target site in intron 11 (Figure 5.1). As no repair template was provided, CRISPR/Cas9-mediated DSBs are favourable repaired by the error prone NHEJ pathway, which often leaves indel mutations. To assess the frequency of indel mutations and consequently the cutting efficiency of the gRNA, a TIDE analysis was undertaken

using the software package designed by Brinkman et al. (2014). To do this, DNA was extracted from pools of CRISPR targeted melanocytes and non-targeted control melanocytes. The two DNA samples were separately sequenced across the gRNA target site. The quantitative trace data from the two Sanger sequencing reaction was then used by the TIDE software to determine the total cutting efficiency of the gRNA to be 33.2% (Figure 4.6a). Previous gRNA efficiencies measured at the WTCHG indicated this was sufficient to progress to gene targeting in mESCs. The TIDE software also generated a quality assurance graph that indicated that aberrations >20% between the control and Fbxw7 targeted sequences originate proximal to the expected gRNA cut site (Within 12 bp; Figure 2.5b).

Figure 5.1 Cloned CRISPR (cCRISPR) expression plasmid. In mammalian cells this plasmid expresses the essential components for CRISPR/Cas9 gene targeting. The CRISPR expressed components are the unique gRNA and SpCas9 mRNA for translation into SpCas9 protein. Within this project, complexing of the gRNA and SpCas9 leads to cleavage at Fbxw7 target site in intron 11.

occurring at significant rates at sites with sequence similarity to the on target sites (Cho et al., 2014; Fu et al., 2013; Hsu et al., 2013). Off target effects could confound phenotypic analyses of the resulting Fbxw7R482Q mouse model. It has been demonstrated that, although each nucleotide within the 20 nt crRNA segment of the gRNA contributes to overall SpCas9 specificity, single mismatches are often well tolerated, and several mismatches can also be tolerated depending on their location within crRNA sequence and the context within the genome (Hsu et al., 2013; Pattanayak et al., 2013). Experimental protocols for screening off target effects are laborious, costly and time consuming, and is not yet a routine procedure for CRISPR/Cas9 GM mouse production. Instead an in silico based approach for determining off target potential of the gRNA was employed in this study. Within the mouse genome (GRCm38/mm10) the designed 20 nt crRNA portion of the gRNA had no additional target sites with 0 or 1 nt mismatches. However, for crRNA mismatches of 2, 3 and 4 nt there were 1, 15 and 125 off-target sites, respectively (Table 4.1). These computational results suggested the gRNA had minimal potential for off target effects in the mouse genome.

The optimisation and validation gRNA design is critical for reducing off target potential, however, alternative methods to improve the specificity of CRISPR/Cas9 have been developed. One strategy is to use different Cas9 systems, such as N. meningitides, which can offer better targeting specificity by virtue of its longer crRNA (24 nt for N. meningitidis) and longer PAM sequence (5’- NNNNGATT for N. meningitides; Hou et al., 2013). Another strategy has been to use a modified version of SpCas9 that can only introduce a single-strand nick at the target site, rather than a DSB. Use of a pair of ‘nickase’ Cas9 complexes with binding sites on opposite strands of the target sequence can produce the equivalent of a DSB with 5’ overhangs. At an off-target site, a single- strand nick would be repaired by a different mechanism, known as the base excision repair pathway, which is much less likely to leave indel mutations. Since the likelihood of two nickases binding nearby each other elsewhere in the genome is very low, the off target mutation rate is greatly reduced. Indeed, testing of this nickase systems in mammalian cells has demonstrated up to a threefold reduction in off target activity with only a slight reduction in on target efficacy (Cho et al., 2014; Ran et al.).

After verifying the efficiency and specificity of the selected gRNA, the study was progressed to gene targeting in mESCs. For this, the cCRISPR expression plasmid and Fbxw7 donor plasmid were electroporated in mESCs. The simultaneous transfection of the cCRISPR expression plasmid and the Fbxw7 donor plasmid was aimed at inducing mESCs to repair the CRISPR/Cas9-mediated DSB via HDR and thus copy the targeting construct into the break site. Figure 5.2 shows a schematic diagram of CRISPR/Cas9-mediated HDR using the Fbxw7 donor plasmid as a template. As the transfected cCRISPR expression plasmid contained a puromycin resistance gene, and the Fbxw7 donor plasmid was designed with a neomycin resistance gene and diphtheria toxin A cassette located outside the homology arms, this allowed for both positive and negative selection in transfected mESCs. After eight days of selection, 22 mESC clones were picked and expanded. Of the 22 clones, 4 were confirmed for 5’ and 3’ construct integration by series of three PCR screening protocols. However, PCR screening approaches cannot detect concurrent random integration and, therefore, full characterization requires Southern blotting (Wang et al., 2015). Although highly recommended, Southern blotting was not performed in this study. This decision was made with the foresight that 2 of the 4 clones will be used for blastocyst microinjection and all established mice lines undergo a complete characterization.

Figure 5.2 CRISPR/Cas9-medaited gene targeting with Fbxw7 donor plasmid. SpCas9 and gRNA are expressed from the cCRISPR expression plasmid. A complex between gRNA and SpCas9 is

The targeting construct used in this study took advantage of a Cre-dependent conditional KI system. Once integrated, this construct design will ultimately allow investigators to study the Fbxw7(R482Q) point mutation in specific cell lineage or at a specific developmental stage. The conditional KI system used in the Fbxw7 donor plasmid is known as the ‘minigene’ method (Figure 1.6b). The minigene method in the conditional KI construct contains a WT Fbxw7 minigene (an intronless gene segment that contains exon 12, 13 and 14) and a STOP cassette, which are collectively floxed. Directly downstream from the floxed minigene system is the mutant Fbxw7(R482Q) allele and a CFP reporter gene. In the presence of Cre recombinase, recombination between the loxP sites will occur, resulting in excision of the floxed 3.5kb DNA sequence containing the WT Fbxw7 minigene and STOP cassette (Figure 5.3). Following recombination of the loxP sites, the mutant Fbxw7(R482Q) allele and a CFP report gene will be expressed from their natural promoter. By including the CFP reporter gene in the construct it allows scientists to visually identify cells that are expressing the conditional mutant Fbxw7(R482Q) allele.

Figure 5.3 Fbxw7 conditional knock-in construct, with and without Cre recombination. Recombination of the loxP sites results in 3553 bp deletion, which includes the WT Fbxw7 minigene and STOP cassette that prevent expression of the mutant Fbxw7(R482Q) and CFP reporter gene.

To test the functionality of the conditional KI system in the four positive mESC clones, TAT-Cre recombinase was added to cells at 40% confluent. Once confluent, TAT-Cre treated clones were viewed under an epifluorescence microscope using a DAPI filter to visualise CFP expression. The images obtained from the microscopy session showed faint evidence of increased fluorescence

from CFP expression in TAT-Cre treated cells (Figure 4.14). To improve these findings, optimization of the TAT-Cre treatment protocol may increase CFP expression. Additionally, the use of a confocal fluorescence microscope as opposed to a standard epifluorescence microscope may allow for easier detection of CFP. As the microscopy session offered inclusive results, the functionality of the conditional KI system in the TAT-Cre treated clones required further investigation. To do this TAT-Cre clones were lysed and DNA extracted. Using a PCR based approach it was confirmed that the predicted 3.5 kb DNA sequence had been deleted from a proportion of cells in all four TAT-Cre treated clones (Figure 4.13). This indicated that Cre- mediated recombination between the loxP sites had occurred. These findings confirmed that the Cre-dependent conditional KI system was functional in all four confirmed mESC clones.

Following successful screening and functional testing, two of the four confirmed mESC clones (D01 and E01) were thawed from frozen copies and prepared for blastocyst microinjection. The positive mESCs were developed on a male ‘black coat’ JM8.F6 mESC background and injected into ‘white coat’ Bl6-albino blastocysts before being transferred into CD1 fosters mothers. Male mESCs were used to favour the development of male offspring, which are beneficial for future breeding. Successful integration of mESCs into the inner cell mass of the blastocysts results in black and white chimeric pups. The desired outcome was to generate germline competent chimeric males for germline transmission. Four litters were produced with a total of 8 chimeric males born that ranged in chimerism from 50 to 95% (Table 4.2). The percentage black coat colour was used to estimate the contribution of the mESC to the mice. However, it should be noted that coat colour does not fully reflect the injected mESCs contribution to the germ cells (Carstea et al., 2009). Currently, two 95% chimeric males are set for mating with white albino females in the hope of generating heterozygous Fbxw7R482Q (F1) offspring (Figure 5.4). Germline transmission of the mESC derived genes will result in black pups due to dominant inheritance of the black coat colour. Once a breeding population is established the Tomlinson group at the WTCHG plans to cross the

Fbxw7R482Q _{mice with Villin-Cre mice. Villin-Cre mice express Cre recombinase in villus and crypt}

epithelial cells of the small and large intestines. Crossing of the Fbxw7R482Q mice with the VIllin-

Cre mice will allow the Tomlinson group to study the role of the Fbxw7(R482Q) missense point

Figure 5.4 Breeding scheme to generate heterozygous Fbxw7R482Q (F1) from chimeric males (F0). Future plans are to cross Fbxw7R482Q mice with Villin-Cre mice to study the role of the Fbxw7(R482Q) mutation in the development of colorectal cancer. Image was modified and adapted from Lodish et al. (2000).

6

Conclusion

In summary, the application of CRISPR/Cas9 in mESCs remains the ‘gold standard’ for generating large-scale conditional KI mouse models. This study successfully employed a CRISPR/Cas9- mediated gene targeting approach in mESCs to generate conditional KI chimeric male mice to be used for germline transmission. To limit off target effects and enhance productivity, the CRISPR gRNA used in this study was tested via combination of in vitro and in silico based methods to validate both efficiency and specificity. The construct integrated into CRISPR/Cas9 target site in intron 11 of the Fbxw7 gene was a 5.5 kb Cre-dependent conditional KI that harboured a missense point mutation (Fbxw7(R482Q)) in exon 12. Fbxw7(R482Q) is found in a diverse range of human cancers. The targeting construct also included a CFP reporter gene to track expression of the mutant Fbxw7 allele. Following PCR based screening and functional testing of the targeted mESCs, two confirmed clones were microinjected into WT blastocysts. From the resulting four litters, eight chimeric males were born with up to 95% chimerism based on coat colour. Two 95% chimeric males are currently set for mating WT females in the hope of gaining germline transmission of the mESC derived genes. Successful transmission will generate heterozygous

Fbxw7R482Q

mice. The Tomlinson group at the WTCHG plans to cross the Fbxw7R482Q mouse model with Villin-Cre transgenic mice. This will enable the group to study the role Fbxw7(R482Q) point mutation in development of colorectal cancer.

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