Debugging the genetic code: Non-viral in vivo delivery of therapeutic genome editing technologies

Abstract

Efforts to precisely correct genomic mutations that underlie hereditary diseases for therapeutic benefit have advanced alongside the emergence and improvement of genome engineering technologies. These methods can be divided into two main classes: active nucleasebased platforms including the popular CRISPR/Cas9 system and oligo/polynucleotide strategies including triplex-forming oligonucleotides (TFOs), such as peptide nucleic acids (PNAs). These technologies have been successful in cell culture and in animals, but important challenges remain before these tools can be translated into the clinic; they must be effectively delivered to and taken up by specific cell types of interest, achieve correction levels in target cells that significantly ameliorate the disease phenotype, and demonstrate minimal off-target and toxicity effects. Here we review and compare the current strategies and non-viral delivery methods, mainly lipid and polymeric vehicles, proposed for genome editing of inherited disorders with a focus on in vivo delivery and efficacy. While the path to a safe and effective medical treatment may be arduous, the future outlook of therapeutic genome editing remains promising as long as precise technologies can be combined with efficient delivery.

Chemical synthesis of stimuli-responsive guide RNA for conditional control of CRISPR-Cas9 gene editing

“…We simultaneously assayed for compatibility of chemically modified crRNAs with mRNA delivery of Cas9 using HEK293T cells. 7,8 Learning directly from the success of 3BO-141718-crRNA EMX1 , we chose to introduce BO modifications at G 14 G 17 U 18 in the seed region of the 42-nt crRNA HBB (Figure 4A) to obtain 3BO-141718-crRNA HBB (containing the seed region of ACAGGG BO CAG BO U BO AA). Indeed, 3PS-2′-OMe-141718-crRNA HBB , among several variants with different modification sites, showed comparable activity with the native crRNA HBB (Figure S10).…”

“…[2][3][4] In addition to delivery of DNA that encodes the guide RNA (gRNA) and Cas9 protein using lipid nanoparticles or recombinant viruses, 5,6 efficient genome editing has also been observed with ribonucleoprotein (RNP) and mRNA delivery. 7,8 As RNA usually has a shorter half-life compared to DNA, these delivery methods are often associated with considerably lower off-target effects. 9,10 For this purpose, chemically modified gRNA, [11][12][13][14][15] either in the form of a single guide RNA (sgRNA) 11 or a two-component CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), 12 is actively studied.…”

Recent Advancement and Innovations in CRISPR/Cas and CRISPR Related Technologies: A review

“…Cell-derived membrane vesicles (CMVs): CMVs have been utilized with delivery of siRNA and miNRA delivery into cells for gene therapy as demonstrated by Van Dommelen et al, but literature review does not provide its use with CRISPR/Cas9 methods [87,88]. However, it will be an excellent idea to use this biologically compatible membrane for delivering CRISPR.…”

“…While the detailed insight about molecular understanding of this process is still being deciphered, it seems that the methodology use the inherent cell's DNA repair system and exchange the non-desirable fragment with foreign DNA segment [93]. This methodology could incorporate non-homologous repair end joining or Homology-Dependent Repair (HDR) in combination [88,93]. Currently this technique is rapidly expanding with various new biotechnological innovations and tailoring it to research clinical and research use.…”

“…Various available platforms for such techniques from single stranded oligonucleotide to Triple Forming Oligonucleotides (TFOs). Attempts have been made for direct delivery of exogenous Nucleic Acids (single or double-stranded DNA) with quite a success [88]. "Single Fragment Homologous Replacement (SFHR)", though not a new addition, is a method where a where a very short DNA segment of size 1Kb replaces a homologous region on host DNA.…”