Self-delivering CRISPR RNAs for AAV Co-delivery and Genome Editingin vivo

Abstract

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.

Breaking genetic shackles: The advance of base editing in genetic disorder treatment

“…It has been reported that multiple groups have utilized a dual AAV system to deliver base editing treatments for diseases such as amyotrophic lateral sclerosis ( Lim et al, 2020 ), duchenne muscular dystrophy (DMD) ( Ryu et al, 2018 ; Xu L. et al, 2021 ; Chemello et al, 2021 ), metabolic liver diseases ( Villiger et al, 2018 ), hutchinson-gilford progeria syndrome ( Koblan et al, 2021b ), and hearing loss (HL) ( Yeh et al, 2020 ) in mouse models. In addition, researchers have developed smaller Cas nucleases to enable single AAV vector delivery ( Chen et al, 2022 ; Kweon et al, 2023 ) and strategies to reduce the long-term expression of edited genes via AAV delivery ( Ibraheim et al, 2021 ; Zhang H. et al, 2023 ). Despite these advancements, the clinical application of AAV vectors still faces certain limitations, with pre-existing immunity against AAV being a major challenge in AAV gene therapy ( Kruzik et al, 2019 ).…”