CRISPR-derived genome editing therapies: Progress from bench to bedside

Gene Editing Technologies to Target HBV cccDNA

“…Ribonucleoprotein (RNP) or protein complexes can also be formulated directly into lipid nanoparticles (LNPs) [47]. One of the advantages of this approach, with respect to nuclease mRNA and gRNA LNPs, is the adjusted 1:1 stoichiometry of the editor/gRNA complex [18]; the challenge is to keep the complex active, which can be difficult in terms of manufacturing, as well as the optimization of the protein-LNP formulation for in vivo targeting [44]. RNP LNP delivery has enabled efficient editing in vitro for a number of applications, in particular, for inhibiting HBV replication in infected HepG2-NTCP cells [47].…”

“…Off-target effects are unintended genetic modifications that occur through the use of gene editing technology. Off-target effects can be gRNA-dependent (i.e., associated with the gRNA sequence recognizing DNA) or -independent (related to the alternative activities of the gene editing enzyme) [18].…”

“…Aside from the requirement of a PAM sequence, Cas9 is easily targetable to new sites simply by changing the gRNA sequence. As with other nucleases, CRISPR/Cassystem-induced DSBs can be processed by NHEJ to generate indels or by HDR to insert a sequence defined by an exogenous DNA template [13,[16][17][18] (Table 1). Base editors (BEs) are alternative gene editing tools that introduce precise mutations in targeted sequences through chemical reactions of deamination without inducing DSBs, thus not requiring donor DNA templates for gene correction.…”

“…Furthermore, most CBEs contain a uracil glycosylase inhibitor peptide (UGI) that prevents the activation of the base excision repair system. As base editors do not generate DSBs and therefore do not stimulate NHEJ, they generate fewer off-target indel mutations and are more efficient in non-dividing cells compared to the standard nucleases [17][18][19][20]. Glycosylase base editors have been developed more recently through the replacement of UGI with a uracil-DNA glycosylase (UNG) in certain CBEs, which has enabled C>A transversion changes in Escherichia coli and C>G transversion changes in mammalian cells [21].…”

Gene editing is suitable to treat GM1 Gangliosidosis: a proof-of-concept study

“…To date, BE has the best efficiency rate compared to PE or CRISPR-Cas9 coupled to a donor. However, only C-to-T (CBE) and A-to-G (ABE) base editing methods are fully validated for gene therapy 12,13 . Moreover, CBE and ABE are active on a wide editing window (max efficiency nucleotide 4 to 6 from the 5' end of the sgRNA depending on the BE version), suggesting that bystander C or A nucleotides should be also modified into T or G within this editing window (bystander edits) 14,15 .…”