RESUMO
Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a 'protecting oligo'), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNA in vivo. Finally, we achieved in vivo genome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation.
Assuntos
Edição de Genes , RNA Guia de Sistemas CRISPR-Cas , Animais , Camundongos , Distribuição Tecidual , RNA/genética , OligonucleotídeosRESUMO
Simple 2'-OMe-chemical modification in the loop region of the 15mer G-rich DNA sequence GGTTGGTGTGGTTGG is reported. The G-quadruplex structure of this thrombin-binding aptamer (TBA), is stabilized by single modifications (Tâ¯ââ¯2'-OMe-U), depending on the position of the modification. The structural stability also renders significantly increased inhibition of thrombin-induced fibrin polymerization, a process closely associated with blood-clotting.
Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Trombina/antagonistas & inibidores , Aptâmeros de Nucleotídeos/química , Sítios de Ligação , Coagulação Sanguínea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fibrina/antagonistas & inibidores , Fibrina/metabolismo , Quadruplex G , Estrutura Molecular , Polimerização/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
An antiparallel, functional RNA G-quadruplex of the 2'-5'-linked thrombin-binding aptamer (iso-rTBA) is reported for the first time. It can inhibit clotting and is remarkably stable to nuclease-degradation, besides having high thermal stability. It is thus, a superior candidate to TBA, rTBA or isoTBA, for further development as an anticoagulant.