TY - JOUR
T1 - Harnessing Peptide Nucleic Acids and the Eukaryotic Resolvase MOC1 for Programmable, Precise Generation of Double-Strand DNA Breaks
AU - Sivakrishna Rao, Gundra
AU - Saleh, Ahmed H.
AU - Melliti, Firdaws
AU - Muntjeeb, Syed
AU - Mahfouz, Magdy
N1 - Publisher Copyright:
© 2024 The Authors. Published by American Chemical Society.
PY - 2024/2/13
Y1 - 2024/2/13
N2 - Programmable site-specific nucleases (SSNs) hold extraordinary promise to unlock myriad gene editing applications in medicine and agriculture. However, developing small and specific SSNs is needed to overcome the delivery and specificity translational challenges of current genome engineering technologies. Structure-guided nucleases have been harnessed to generate double-strand DNA breaks but with limited success and translational potential. Here, we harnessed the power of peptide nucleic acids (PNAs) for site-specific DNA invasion and the generation of localized DNA structures that are recognized and cleaved by the eukaryotic resolvase AtMOC1 from Arabidopsis thaliana. We named this technology PNA-assisted Resolvase-mediated (PNR) editing. We tested the PNR editing concept in vitro and demonstrated its precise target specificity, examined the nucleotide requirement around the PNA invasion for the AtMOC1-mediated cleavage, mapped the AtMOC1-mediated cleavage sites, tested the role of different types and lengths of PNA molecules invasion into dsDNA for the AtMOC1-mediated cleavage, optimized the in vitro PNA invasion and AtMOC1 cleavage conditions such as temperature, buffer conditions, and cleavage time points, and demonstrated the multiplex cleavage for precise fragment release. We discuss the best design parameters for efficient, site-specific in vitro cleavage using PNR editors.
AB - Programmable site-specific nucleases (SSNs) hold extraordinary promise to unlock myriad gene editing applications in medicine and agriculture. However, developing small and specific SSNs is needed to overcome the delivery and specificity translational challenges of current genome engineering technologies. Structure-guided nucleases have been harnessed to generate double-strand DNA breaks but with limited success and translational potential. Here, we harnessed the power of peptide nucleic acids (PNAs) for site-specific DNA invasion and the generation of localized DNA structures that are recognized and cleaved by the eukaryotic resolvase AtMOC1 from Arabidopsis thaliana. We named this technology PNA-assisted Resolvase-mediated (PNR) editing. We tested the PNR editing concept in vitro and demonstrated its precise target specificity, examined the nucleotide requirement around the PNA invasion for the AtMOC1-mediated cleavage, mapped the AtMOC1-mediated cleavage sites, tested the role of different types and lengths of PNA molecules invasion into dsDNA for the AtMOC1-mediated cleavage, optimized the in vitro PNA invasion and AtMOC1 cleavage conditions such as temperature, buffer conditions, and cleavage time points, and demonstrated the multiplex cleavage for precise fragment release. We discuss the best design parameters for efficient, site-specific in vitro cleavage using PNR editors.
UR - http://www.scopus.com/inward/record.url?scp=85184801721&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.3c05133
DO - 10.1021/acs.analchem.3c05133
M3 - Article
C2 - 38300270
AN - SCOPUS:85184801721
SN - 0003-2700
VL - 96
SP - 2599
EP - 2609
JO - ANALYTICAL CHEMISTRY
JF - ANALYTICAL CHEMISTRY
IS - 6
ER -