Mutant allele-specific CRISPR disruption in DYT1 dystonia fibroblasts restores cell function
Most individuals affected with DYT1 dystonia have a heterozygous 3-bp deletion in the TOR1A gene (c.907_909delGAG). The mutation appears to act through a dominant-negative mechanism compromising normal torsinA function, and it is proposed that reducing mutant torsinA may normalize torsinA activity....
| Egile Nagusiak: | , , , , , , , , , , , , |
|---|---|
| Formatua: | Artikulua |
| Hizkuntza: | English |
| Argitaratua: |
Elsevier
2020
|
| Sarrera elektronikoa: | http://psasir.upm.edu.my/id/eprint/86867/1/Mutant%20allele.pdf |
| _version_ | 1825952278086942720 |
|---|---|
| author | Cruz, Lilian Bence, György Cheah, Pike See Kleinstiver, Benjamin P. Eimer, William A. Garcia, Sara P. Sharma, Nutan Ozelius, Laurie J. Cristopher, Bragg D. Joung, Keith J. Norberto de Souza, Osmar Macedo Timmers, Luis Fernando Saraiva Breakefield, Xandra O. |
| author_facet | Cruz, Lilian Bence, György Cheah, Pike See Kleinstiver, Benjamin P. Eimer, William A. Garcia, Sara P. Sharma, Nutan Ozelius, Laurie J. Cristopher, Bragg D. Joung, Keith J. Norberto de Souza, Osmar Macedo Timmers, Luis Fernando Saraiva Breakefield, Xandra O. |
| author_sort | Cruz, Lilian |
| collection | UPM |
| description | Most individuals affected with DYT1 dystonia have a heterozygous 3-bp deletion in the TOR1A gene (c.907_909delGAG). The mutation appears to act through a dominant-negative mechanism compromising normal torsinA function, and it is proposed that reducing mutant torsinA may normalize torsinA activity. In this study, we used an engineered Cas9 variant from Streptococcus pyogenes (SpCas9-VRQR) to target the mutation in the TOR1A gene in order to disrupt mutant torsinA in DYT1 patient fibroblasts. Selective targeting of the DYT1 allele was highly efficient with most common non-homologous end joining (NHEJ) edits, leading to a predicted premature stop codon with loss of the torsinA C terminus (delta 302–332 aa). Structural analysis predicted a functionally inactive status of this truncated torsinA due to the loss of residues associated with ATPase activity and binding to LULL1. Immunoblotting showed a reduction of the torsinA protein level in Cas9-edited DYT1 fibroblasts, and a functional assay using HSV infection indicated a phenotypic recovery toward that observed in control fibroblasts. These findings suggest that the selective disruption of the mutant TOR1A allele using CRISPR-Cas9 inactivates mutant torsinA, allowing the remaining wild-type torsinA to exert normal function. |
| first_indexed | 2024-03-06T10:42:32Z |
| format | Article |
| id | upm.eprints-86867 |
| institution | Universiti Putra Malaysia |
| language | English |
| last_indexed | 2024-03-06T10:42:32Z |
| publishDate | 2020 |
| publisher | Elsevier |
| record_format | dspace |
| spelling | upm.eprints-868672021-12-29T04:10:32Z http://psasir.upm.edu.my/id/eprint/86867/ Mutant allele-specific CRISPR disruption in DYT1 dystonia fibroblasts restores cell function Cruz, Lilian Bence, György Cheah, Pike See Kleinstiver, Benjamin P. Eimer, William A. Garcia, Sara P. Sharma, Nutan Ozelius, Laurie J. Cristopher, Bragg D. Joung, Keith J. Norberto de Souza, Osmar Macedo Timmers, Luis Fernando Saraiva Breakefield, Xandra O. Most individuals affected with DYT1 dystonia have a heterozygous 3-bp deletion in the TOR1A gene (c.907_909delGAG). The mutation appears to act through a dominant-negative mechanism compromising normal torsinA function, and it is proposed that reducing mutant torsinA may normalize torsinA activity. In this study, we used an engineered Cas9 variant from Streptococcus pyogenes (SpCas9-VRQR) to target the mutation in the TOR1A gene in order to disrupt mutant torsinA in DYT1 patient fibroblasts. Selective targeting of the DYT1 allele was highly efficient with most common non-homologous end joining (NHEJ) edits, leading to a predicted premature stop codon with loss of the torsinA C terminus (delta 302–332 aa). Structural analysis predicted a functionally inactive status of this truncated torsinA due to the loss of residues associated with ATPase activity and binding to LULL1. Immunoblotting showed a reduction of the torsinA protein level in Cas9-edited DYT1 fibroblasts, and a functional assay using HSV infection indicated a phenotypic recovery toward that observed in control fibroblasts. These findings suggest that the selective disruption of the mutant TOR1A allele using CRISPR-Cas9 inactivates mutant torsinA, allowing the remaining wild-type torsinA to exert normal function. Elsevier 2020-09-04 Article PeerReviewed text en http://psasir.upm.edu.my/id/eprint/86867/1/Mutant%20allele.pdf Cruz, Lilian and Bence, György and Cheah, Pike See and Kleinstiver, Benjamin P. and Eimer, William A. and Garcia, Sara P. and Sharma, Nutan and Ozelius, Laurie J. and Cristopher, Bragg D. and Joung, Keith J. and Norberto de Souza, Osmar and Macedo Timmers, Luis Fernando Saraiva and Breakefield, Xandra O. (2020) Mutant allele-specific CRISPR disruption in DYT1 dystonia fibroblasts restores cell function. Molecular Therapy-Nucleic Acids, 21. pp. 1-12. ISSN 2162-2531 https://www.sciencedirect.com/science/article/pii/S2162253120301359 10.1016/j.omtn.2020.05.009 |
| spellingShingle | Cruz, Lilian Bence, György Cheah, Pike See Kleinstiver, Benjamin P. Eimer, William A. Garcia, Sara P. Sharma, Nutan Ozelius, Laurie J. Cristopher, Bragg D. Joung, Keith J. Norberto de Souza, Osmar Macedo Timmers, Luis Fernando Saraiva Breakefield, Xandra O. Mutant allele-specific CRISPR disruption in DYT1 dystonia fibroblasts restores cell function |
| title | Mutant allele-specific CRISPR disruption in DYT1 dystonia fibroblasts restores cell function |
| title_full | Mutant allele-specific CRISPR disruption in DYT1 dystonia fibroblasts restores cell function |
| title_fullStr | Mutant allele-specific CRISPR disruption in DYT1 dystonia fibroblasts restores cell function |
| title_full_unstemmed | Mutant allele-specific CRISPR disruption in DYT1 dystonia fibroblasts restores cell function |
| title_short | Mutant allele-specific CRISPR disruption in DYT1 dystonia fibroblasts restores cell function |
| title_sort | mutant allele specific crispr disruption in dyt1 dystonia fibroblasts restores cell function |
| url | http://psasir.upm.edu.my/id/eprint/86867/1/Mutant%20allele.pdf |
| work_keys_str_mv | AT cruzlilian mutantallelespecificcrisprdisruptionindyt1dystoniafibroblastsrestorescellfunction AT bencegyorgy mutantallelespecificcrisprdisruptionindyt1dystoniafibroblastsrestorescellfunction AT cheahpikesee mutantallelespecificcrisprdisruptionindyt1dystoniafibroblastsrestorescellfunction AT kleinstiverbenjaminp mutantallelespecificcrisprdisruptionindyt1dystoniafibroblastsrestorescellfunction AT eimerwilliama mutantallelespecificcrisprdisruptionindyt1dystoniafibroblastsrestorescellfunction AT garciasarap mutantallelespecificcrisprdisruptionindyt1dystoniafibroblastsrestorescellfunction AT sharmanutan mutantallelespecificcrisprdisruptionindyt1dystoniafibroblastsrestorescellfunction AT ozeliuslauriej mutantallelespecificcrisprdisruptionindyt1dystoniafibroblastsrestorescellfunction AT cristopherbraggd mutantallelespecificcrisprdisruptionindyt1dystoniafibroblastsrestorescellfunction AT joungkeithj mutantallelespecificcrisprdisruptionindyt1dystoniafibroblastsrestorescellfunction AT norbertodesouzaosmar mutantallelespecificcrisprdisruptionindyt1dystoniafibroblastsrestorescellfunction AT macedotimmersluisfernandosaraiva mutantallelespecificcrisprdisruptionindyt1dystoniafibroblastsrestorescellfunction AT breakefieldxandrao mutantallelespecificcrisprdisruptionindyt1dystoniafibroblastsrestorescellfunction |