Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches

Abstract

Since its discovery in 2012, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system has supposed a promising panorama for developing novel and highly precise genome editing-based gene therapy (GT) alternatives, leading to overcoming the challenges associated with classical GT. Classical GT aims to deliver transgenes to the cells via their random integration in the genome or episomal persistence into the nucleus through lentivirus (LV) or adeno-associated virus (AAV), respectively. Although high transgene expression efficiency is achieved by using either LV or AAV, their nature can result in severe side effects in humans. For instance, an LV (NCT03852498)- and AAV9 (NCT05514249)-based GT clinical trials for treating X-linked adrenoleukodystrophy and Duchenne Muscular Dystrophy showed the development of myelodysplastic syndrome and patient's death, respectively. In contrast with classical GT, the CRISPR/Cas9-based genome editing requires the homologous direct repair (HDR) machinery of the cells for inserting the transgene in specific regions of the genome. This sophisticated and well-regulated process is limited in the cell cycle of mammalian cells, and in turn, the nonhomologous end-joining (NHEJ) predominates. Consequently, seeking approaches to increase HDR efficiency over NHEJ is crucial. This manuscript comprehensively reviews the current alternatives for improving the HDR for CRISPR/Cas9-based GTs.

Keywords: CRISPR/Cas9; HDR; NHEJ; genome editing.

Figure 1

Cas9 protein structure and Cas9-mediated DNA double-strand breaks. In (a), the full Cas9 protein structure isolated from Streptococcus pyogenes is shown. Notice the recognizing (REC) and nuclease (NUC) lobes, which mediate the targeted DNA recognition and double-strand break, respectively. The arginine (Arg) 1333 and 1335 residues responsible for PAM sequence recognition are also displayed. In (b), a classical overview of the ribonucleoprotein (RNP) complex (Cas9 together with sgRNA) interacting with the targeted DNA is shown. The histidine 840 (H840) catalyzes the break of the sgRNA interacting DNA strand, while aspartate 10 (D10) mediates breaking on the opposite strand. Note that the RuvC endonuclease domain comprises three segments: RuvC I, II, and III, while the HNH catalytic domain comprises only one segment. Likewise, three alpha-helical domains (HD) at the REC lobe are placed and primarily responsible for nucleic acid binding. Finally, L-I and L-II are key linkers that aid the connection between RuvC and HNH. CTD: Carboxy-terminal domain. This figure was created with BioRender.com.

Figure 2

Major steps in non-homologous end joining (NHEJ) and Homology-directed repair (HDR). NHEJ is initiated by binding of Ku to the DSB ends. This is followed by recruiting DNA-PKcs and other necessary scaffolding factors that bring DSB ends together. Nucleases process the incompatible ends, polymerases fill gaps, and finally, the ends are ligated by DNA ligases. This process ultimately leads to the formation of INDELs. HDR is initiated by the binding of MRN that stabilizes DSB ends. Then, 5′ exonucleases produce short-range resections followed by long-range resections. The long 3′ overhangs are directed for homology search and strand invasion by RAD51, leading to D-loops’ formation. Finally, the exchanged DNA strands are resolved, leading to precise editing. Several platforms can be used as donor templates, including long single-stranded oligodeoxynucleotide (lssDNA), single-stranded oligodeoxynucleotide (ssODN), double-stranded oligodeoxynucleotide (dsODN), and double-stranded DNA (dsDNA) which can be carried through non-viral vectors, as well as DNA templates loaded into viral vectors. For more details related to donor templates and their impact on HDR, we strongly encourage readers to review the paper by Shakirova et al., 2023 [52]. Although differences in the recognition of ssDNA (i.e., BRCA1) and dsDNA (i.e., BRCA2) have been well-documented, in both scenarios, the homologous recombination takes place, leading to precise genome editing in the context of the CRISPR/Cas9 system. This figure was created with BioRender.com.

Figure 3

Strategies for increasing the knock-in efficiency in CRISPR/Cas9-based genome editing. Increasing CRISPR/Cas9-mediated genome editing events involves factors such as the type of cell (a), as well as indirect (b) or direct (c) HDR modulation by using several strategies. Several small molecules can also achieve HDR increase, although their full mechanism of action is still to be uncovered (d). This figure was created with BioRender.com.