Most of the times, dominantly inherited genetic diseases cannot be tackled by gene addition-type gene therapies, highlighting the need for specific gene editing approaches. The advent of CRISPR/Cas9 has revolutionized the field of precision medicine, but highly specific and adaptable platforms are still looked out for, especially when focusing on allele-specific genome editing strategies. In this project we aim at engineering CRISPR/Cas proteins to customize Cas target recognition towards specific PAM-creating mutations. As a proof of concept, we identified three TP63 mutations responsible for ectrodactyly-ectodermal dysplasia-cleft (EEC) syndrome that form a common TATG PAM. This syndrome causes several anomalies, with blindness, caused by progressive limbal stem cell deficiency, being the most impactful on patients’ lives. Nowadays, no treatment is available to patients. The TATG PAM shared by the three mutations, can be targeted by the Cas9 orthologue Cpf1-RVR, or Cas12a-RVR, which has been changed to recognize TATV instead of the canonical TTTV bound by the wild-type (WT) Cpf1. Analyzing the crystal structure of the Cpf1-RVR variant, able to recognize the TATV PAM, we identified two amino acids that can be replaced to increase the specificity of the protein towards TATG. This should allow to knock-out the mutated allele leaving the WT untouched. The plasmid carrying the engineered Cas12a was co-transfected with a plasmid with the TP63 mutation (or its WT version) into HEK cells and the editing efficiency was evaluated by TIDE analysis. Comparing the original Cpf1-RVR with the different combinations of modified Cpf1 proteins showed an increase in both cleavage efficiency and discriminating ability between the WT and the mutated allele. The engineered Cpf1 formed INDELS in the mutated sequence up to 80% of the times, while the WT sequence was altered between 5 and 30% of cases. This data is a huge improvement compared to the RVR protein, that edits the mutated allele only up to 60% and the WT allele up to 70%, depending on the TP63 mutation. Given that the three EEC syndrome-causing mutations are very rare, we tried to expand our gene editing toolbox to tackle a broader range of TP63 mutations. Three of the most frequent mutations (R279C, R280C, R304Q) share a common NCTG PAM, that can potentially be targeted by a modified version of the Cas12a. For this purpose, we further engineered our tailor-made Cpf1, inserting three more amino acid changes (to the two we already modified). Unfortunately, the editing efficiency of this Cas variant dropped to about 20%, also losing any discriminating ability between the WT and the mutated allele. We hypothesize that the insertion of 5 total amino acid changes alters protein stability or DNA binding ability, impeding its normal gene editing ability. Further studies are needed to assess the source of these problems. To overcome the limitations of these preliminary results, in which we transfected thousands of plasmids transfected into HEK293T cells, a condition that is distant from a physiological state in which only two copies of a gene are present inside the cell, we established a biallelic cellular model with the WT and the mutated copies of p63 tagged with fluorescent proteins. We transfected these cells with the various engineered Cas12a plasmids and evaluated editing efficiency by flow cytometry, looking for decreasing fluorescence once the Cas has knocked out its target gene. Unexpectedly, editing efficiency dropped and therefore we investigated the reasons for this: we increased the number on nuclear localization signals to favor nuclear import of the protein and looked into protein stability to check if there is a diminished stability due to the inserted modifications.

Nella maggior parte dei casi, malattie genetiche a trasmissione dominante non possono essere trattate mediante terapia genica con inserimento di una copia sana del gene di interesse. La scoperta di CRISPR/Cas9 ha rivoluzionato il campo della medicina di precisione, ma ha anche evidenziato come manchi ancora una piattaforma specifica e adattabile, specialmente per quanto riguarda le strategie di editing allele specifico. Questo progetto ha lo scopo di ingegnerizzare delle proteine Cas per migliorare il riconoscimento del target contro specifiche mutazioni che formano nuove PAM. Come prova di principio abbiamo identificato tre mutazioni nel gene TP63, responsabili per la sindrome ectrodattilia-displasia dell’ectoderma-labbro leporino (sindrome EEC) e che formano una PAM TATG comune. Questa sindrome causa varie anomalie, tra le quali la cecità, dovuta alla progressiva deplezione delle cellule staminali del limbus, e che ha il maggior impatto sulla vita dei pazienti. Ad oggi non sono disponibili trattamenti per questi pazienti. La PAM TATG comune tra le tre mutazioni, può essere colpita dalla Cpf1-RVR, o Cas12a-RVR. Questa proteina è stata modificata per farle riconoscere la PAM TATV al posto della canonica TTTV riconosciuta dalla versione WT. Analizzando la struttura cristallografica della Cpf1-RVR, abbiamo identificato due amino acidi che possono essere modificati per incrementare la specificità contro la PAM TATG. Ciò dovrebbe permettere di fare un knock-out dell’allele mutate, lasciando quello WT inalterato. Plasmidi con la Cas12a ingegnerizzata sono stati trasfettati assieme ai plasmidi con TP63 mutato (o la sua versione WT) in cellule HEK293T e l’efficienza di editing è stata successivamente valutata mediante analisi TIDE. Comparando la Cpf1-RVR con le diverse varianti da noi prodotte, si può notare un aumento sia nell’efficienza di taglio che nella capacità di discriminazione dei due alleli. Le forme ingegnerizzate formano INDELs fino all’80% dei casi, mentre la sequenza WT è alterata soltanto nel 5-30%. Questi dati sono un incremento significativo rispetto ai dati ottenuti con la Cas12a-RVR, che edita l’allele mutato nel 60% dei casi e l’allele WT fino al 70%, in base alla mutazione presa in considerazione. Dato che le tre mutazioni identificate sono molto rare, abbiamo cercato di espandere il nostro portfolio di Cas12a per poter colpire uno spettro più ampio di mutazioni. Tre delle mutazioni più frequenti (R279C, R280C, R304Q) condividono la PAM NCTG, che può potenzialmente essere targettata da una Cpf1 ulteriormente modificata, inserendo ulteriori tre cambi amminoacidici (oltre ai due già fatti). Inaspettatamente, l’efficienza di taglio della Cas è crollata al 20% e si è persa la capacità di discriminazione allelica. Ipotizziamo che l’inserimento di 5 alterazioni amminoacidiche abbiano alterato la stabilità proteica o la capacità di legame del DNA. Ulteriori studi sono necessari per comprendere queste problematiche. Per superare le limitazioni dei risultati preliminari su plasmidi, in cui abbiamo trasfettato migliaia di copie di plasmidi nelle cellule, situazione molto distante dallo stato fisiologico con due alleli, abbiamo sviluppato un sistema cellulare biallelico contenente una copia WT e una copia con una mutazione di p63, entrambe coniugate con dei fluorofori. Trasfettando queste cellule con i plasmidi contenenti le varie Cas, abbiamo misurato l’efficienza di editing mediante citofluorimetria, guardando la diminuzione del segnale fluorescente dovuto al knock out del gene target. Inaspettatamente, l’efficienza di editing è calata quasi a zero, per cui stiamo cercando di capirne il motivo: abbiamo aumentato il numero dei segnali di localizzazione nucleare coniugati alla proteina e abbiamo investigato la stabilità proteica.

Definizione e ingegnerizzazione di nucleasi CRISPR/Cas per editing allele specifico di malattie genetiche dominanti / Alessio Conci , 2024 May 22. 36. ciclo, Anno Accademico 2022/2023.

Definizione e ingegnerizzazione di nucleasi CRISPR/Cas per editing allele specifico di malattie genetiche dominanti

CONCI, ALESSIO
2024

Abstract

Most of the times, dominantly inherited genetic diseases cannot be tackled by gene addition-type gene therapies, highlighting the need for specific gene editing approaches. The advent of CRISPR/Cas9 has revolutionized the field of precision medicine, but highly specific and adaptable platforms are still looked out for, especially when focusing on allele-specific genome editing strategies. In this project we aim at engineering CRISPR/Cas proteins to customize Cas target recognition towards specific PAM-creating mutations. As a proof of concept, we identified three TP63 mutations responsible for ectrodactyly-ectodermal dysplasia-cleft (EEC) syndrome that form a common TATG PAM. This syndrome causes several anomalies, with blindness, caused by progressive limbal stem cell deficiency, being the most impactful on patients’ lives. Nowadays, no treatment is available to patients. The TATG PAM shared by the three mutations, can be targeted by the Cas9 orthologue Cpf1-RVR, or Cas12a-RVR, which has been changed to recognize TATV instead of the canonical TTTV bound by the wild-type (WT) Cpf1. Analyzing the crystal structure of the Cpf1-RVR variant, able to recognize the TATV PAM, we identified two amino acids that can be replaced to increase the specificity of the protein towards TATG. This should allow to knock-out the mutated allele leaving the WT untouched. The plasmid carrying the engineered Cas12a was co-transfected with a plasmid with the TP63 mutation (or its WT version) into HEK cells and the editing efficiency was evaluated by TIDE analysis. Comparing the original Cpf1-RVR with the different combinations of modified Cpf1 proteins showed an increase in both cleavage efficiency and discriminating ability between the WT and the mutated allele. The engineered Cpf1 formed INDELS in the mutated sequence up to 80% of the times, while the WT sequence was altered between 5 and 30% of cases. This data is a huge improvement compared to the RVR protein, that edits the mutated allele only up to 60% and the WT allele up to 70%, depending on the TP63 mutation. Given that the three EEC syndrome-causing mutations are very rare, we tried to expand our gene editing toolbox to tackle a broader range of TP63 mutations. Three of the most frequent mutations (R279C, R280C, R304Q) share a common NCTG PAM, that can potentially be targeted by a modified version of the Cas12a. For this purpose, we further engineered our tailor-made Cpf1, inserting three more amino acid changes (to the two we already modified). Unfortunately, the editing efficiency of this Cas variant dropped to about 20%, also losing any discriminating ability between the WT and the mutated allele. We hypothesize that the insertion of 5 total amino acid changes alters protein stability or DNA binding ability, impeding its normal gene editing ability. Further studies are needed to assess the source of these problems. To overcome the limitations of these preliminary results, in which we transfected thousands of plasmids transfected into HEK293T cells, a condition that is distant from a physiological state in which only two copies of a gene are present inside the cell, we established a biallelic cellular model with the WT and the mutated copies of p63 tagged with fluorescent proteins. We transfected these cells with the various engineered Cas12a plasmids and evaluated editing efficiency by flow cytometry, looking for decreasing fluorescence once the Cas has knocked out its target gene. Unexpectedly, editing efficiency dropped and therefore we investigated the reasons for this: we increased the number on nuclear localization signals to favor nuclear import of the protein and looked into protein stability to check if there is a diminished stability due to the inserted modifications.
Defining and engineering CRISPR/Cas nucleases for allele-specific editing of dominant genetic diseases
22-mag-2024
DE LUCA, Michele
DE ROSA, LAURA
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