التفاصيل البيبلوغرافية
| العنوان: |
T4 DNA polymerase prevents deleterious on-target DNA damage and enhances precise CRISPR editing. |
| المؤلفون: |
Yang, Qiaoyan, Abebe, Jonathan S, Mai, Michelle, Rudy, Gabriella, Kim, Sang Y, Devinsky, Orrin, Long, Chengzu |
| المصدر: |
EMBO Journal; Sep2024, Vol. 43 Issue 17, p3733-3751, 19p |
| مصطلحات موضوعية: |
CHROMOSOMAL translocation, FRAMESHIFT mutation, DNA polymerases, GENETIC variation, BACTERIOPHAGE T4, GENOME editing |
| مستخلص: |
Unintended on-target chromosomal alterations induced by CRISPR/Cas9 in mammalian cells are common, particularly large deletions and chromosomal translocations, and present a safety challenge for genome editing. Thus, there is still an unmet need to develop safer and more efficient editing tools. We screened diverse DNA polymerases of distinct origins and identified a T4 DNA polymerase derived from phage T4 that strongly prevents undesired on-target damage while increasing the proportion of precise 1- to 2-base-pair insertions generated during CRISPR/Cas9 editing (termed CasPlus). CasPlus induced substantially fewer on-target large deletions while increasing the efficiency of correcting common frameshift mutations in DMD and restored higher level of dystrophin expression than Cas9-alone in human cardiomyocytes. Moreover, CasPlus greatly reduced the frequency of on-target large deletions during mouse germline editing. In multiplexed guide RNAs mediating gene editing, CasPlus repressed chromosomal translocations while maintaining gene disruption efficiency that was higher or comparable to Cas9 in primary human T cells. Therefore, CasPlus offers a safer and more efficient gene editing strategy to treat pathogenic variants or to introduce genetic modifications in human applications. Synopsis: While off-target mutations have been carefully addressed, CRISPR/Cas9 mediated genome editing often causes harmful on-target chromosomal alterations. CasPlus combines Cas9 genome editing with an engineered phage T4 DNA polymerase and reduces the incidence of large deletions and chromosomal translocations. CasPlus can be applied to correct disease-causing mutations such as in Duchenne muscular dystrophy (DMD), but also for generating engineered T cells for cell therapy. CasPlus editing inhibits undesired on-target large deletions while increasing the proportion of precise 1- to 2-base-pair insertions generated during CRISPR/Cas9 editing. CasPlus efficiently corrects the frameshift mutations in DMD (exon 52 deletions) with fewer on-target DNA damage. CasPlus greatly reduces the frequency of on-target large deletions in mouse germline editing. CasPlus represses chromosomal translocation while maintaining gene disruption efficiency that is higher or comparable to Cas9 in multiplex gene-edited human primary T cells. CasPlus combines Cas9 genome editing with an engineered phage T4 DNA polymerase and reduces the incidence of large deletions and chromosomal translocations. [ABSTRACT FROM AUTHOR] |
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| قاعدة البيانات: |
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