One of the major limits of current therapies against cancer and viral infections is the nonspecific toxicity that they often cause on healthy tissues because of their impact on important cellular mechanisms shared, to different extents, between diseased and healthy cells. For this reason, there is an unmet need for more specific and more effective therapies. Wherefore, the aim of my project is the development of a novel strategy, with potential for therapy, that allows the induction of sequence-specific DNA lesions (DNA double-strand break, DSB), by the use of the CRISPR/Cas9 system targeting a genome sequence abnormality in diseased cells, while sparing normal cells. Potential applications of this approach can be cancer cells carrying genomic mutations or chromosomal rearrangements and infected cells carrying an integrated proviral genome. Importantly, whether the aberrant genome sequences are expressed or not is irrelevant for the efficacy of this approach. As a proof of principle, I generated, in two parallel cell systems, an isogenic pair of cell lines with a healthy and a diseased counterpart. The “diseased” target sequence is an integrated proviral genome. To generate them, I infected HeLa and RKO cells with a lentiviral vector containing the sequence of the green fluorescent protein (GFP). I then treated these two cell systems with the purpose of inducing a DSB by retroviral transduction of the Cas9 endonuclease and its RNA guide targeting the integrated GFP sequences. As a negative control, I treated these cell lines in parallel with a Cas9 carrying a scramble guide that does not recognize any sequence in the human genome. Upon these treatments, I observed a preferential reduction of proliferation and an increased mortality in cells bearing the target sequence and transduced with the targeting RNA guide compared to cells without the target sequence or transduced with the scramble guide. I also observed that Cas9-mediated DNA damage is associated with the formation of micronuclei which often stain positive for cGAS and activate an inflammatory response. These results suggest the possibility to “weaponize” the CRISPR/Cas9 system for the elimination of cells with an aberrant genome. However, cells can survive DNA damage insults by repairing them. In order to address this mechanism of “resistance” to the treatment, I investigated if the generation of a sequencespecific DSB can be combined with the inhibition of its repair. Indeed, Cas9-induced DNA damage and inhibition of DNA repair by non-homologous end-joining (NHEJ) by the use of a pharmacological inhibitor of the DNA-dependent protein kinase (DNA-PK), a DNA repair factor involved in NHEJ, further kill target cells. However, DNA-PK inhibition lacks sequence specificity in its activity, thus impacting on the repair of endogenous DNA damage too. For this reason, a sequence-specific DSB repair inhibitor would be desirable. Our group has previously demonstrated that DSBs trigger the recruitment of RNA polymerase II that generates damage-induced long non-coding RNAs (dilncRNAs) at DSB. DilncRNAs are the precursors of small non-coding RNAs called DNA damage response RNAs (DDRNAs) and the interaction between dilncRNAs and DDRNAs is necessary for the recruitment of the proteins involved in DDR, including DNA repair. Noteworthy, antisense oligonucleotides (ASO) against these damage-induced RNA species impair their functions and inhibit the assembly of DDR factors in the form of foci and thus they are effective sequence-specific DNA repair inhibitors. In cells treated with Cas9, I observed a reduction in DDR foci, compared to controls, upon treatment with sequence-specific ASO, confirming the efficacy of ASO also in my experimental system.
WEAPONIZING CRISPR/CAS9 / S. Tavella ; internal advisor: D. Branzei ; tutor: F. D'Adda di Fagagna ; phd coordinator: S. Minucci. Università degli Studi di Milano, 2022 Mar 14. 33. ciclo, Anno Accademico 2021.
WEAPONIZING CRISPR/CAS9
S. Tavella
2022
Abstract
One of the major limits of current therapies against cancer and viral infections is the nonspecific toxicity that they often cause on healthy tissues because of their impact on important cellular mechanisms shared, to different extents, between diseased and healthy cells. For this reason, there is an unmet need for more specific and more effective therapies. Wherefore, the aim of my project is the development of a novel strategy, with potential for therapy, that allows the induction of sequence-specific DNA lesions (DNA double-strand break, DSB), by the use of the CRISPR/Cas9 system targeting a genome sequence abnormality in diseased cells, while sparing normal cells. Potential applications of this approach can be cancer cells carrying genomic mutations or chromosomal rearrangements and infected cells carrying an integrated proviral genome. Importantly, whether the aberrant genome sequences are expressed or not is irrelevant for the efficacy of this approach. As a proof of principle, I generated, in two parallel cell systems, an isogenic pair of cell lines with a healthy and a diseased counterpart. The “diseased” target sequence is an integrated proviral genome. To generate them, I infected HeLa and RKO cells with a lentiviral vector containing the sequence of the green fluorescent protein (GFP). I then treated these two cell systems with the purpose of inducing a DSB by retroviral transduction of the Cas9 endonuclease and its RNA guide targeting the integrated GFP sequences. As a negative control, I treated these cell lines in parallel with a Cas9 carrying a scramble guide that does not recognize any sequence in the human genome. Upon these treatments, I observed a preferential reduction of proliferation and an increased mortality in cells bearing the target sequence and transduced with the targeting RNA guide compared to cells without the target sequence or transduced with the scramble guide. I also observed that Cas9-mediated DNA damage is associated with the formation of micronuclei which often stain positive for cGAS and activate an inflammatory response. These results suggest the possibility to “weaponize” the CRISPR/Cas9 system for the elimination of cells with an aberrant genome. However, cells can survive DNA damage insults by repairing them. In order to address this mechanism of “resistance” to the treatment, I investigated if the generation of a sequencespecific DSB can be combined with the inhibition of its repair. Indeed, Cas9-induced DNA damage and inhibition of DNA repair by non-homologous end-joining (NHEJ) by the use of a pharmacological inhibitor of the DNA-dependent protein kinase (DNA-PK), a DNA repair factor involved in NHEJ, further kill target cells. However, DNA-PK inhibition lacks sequence specificity in its activity, thus impacting on the repair of endogenous DNA damage too. For this reason, a sequence-specific DSB repair inhibitor would be desirable. Our group has previously demonstrated that DSBs trigger the recruitment of RNA polymerase II that generates damage-induced long non-coding RNAs (dilncRNAs) at DSB. DilncRNAs are the precursors of small non-coding RNAs called DNA damage response RNAs (DDRNAs) and the interaction between dilncRNAs and DDRNAs is necessary for the recruitment of the proteins involved in DDR, including DNA repair. Noteworthy, antisense oligonucleotides (ASO) against these damage-induced RNA species impair their functions and inhibit the assembly of DDR factors in the form of foci and thus they are effective sequence-specific DNA repair inhibitors. In cells treated with Cas9, I observed a reduction in DDR foci, compared to controls, upon treatment with sequence-specific ASO, confirming the efficacy of ASO also in my experimental system.File | Dimensione | Formato | |
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