Strategies for precise gene edits in mammalian cells

Abstract

CRISPR-Cas technologies have the potential to revolutionize genetic medicine. However, work is still needed to make this technology clinically efficient for gene correction. A barrier to making precise genetic edits in the human genome is controlling how CRISPR-Cas-induced DNA breaks are repaired by the cell. Since error-prone non-homologous end-joining is often the preferred cellular repair pathway, CRISPR-Cas-induced breaks often result in gene disruption. Homology-directed repair (HDR) makes precise genetic changes and is the clinically desired pathway, but this repair pathway requires a homology donor template and cycling cells. Newer editing strategies, such as base and prime editing, can affect precise repair for relatively small edits without requiring HDR and circumvent cell cycle dependence. However, these technologies have limitations in the extent of genetic editing and require the delivery of bulky cargo. Here, we discuss the pros and cons of precise gene correction using CRISPR-Cas-induced HDR, as well as base and prime editing for repairing small mutations. Finally, we consider emerging new technologies, such as recombination and transposases, which can circumvent both cell cycle and cellular DNA repair dependence for editing the genome.

Keywords: CRISPR-Cas9; MT: RNA/DNA Editing; base editing; gene editing; homology-directed repair; prime editing.

Graphical abstract

Figure 1

DNA repair pathways Comparison of the molecular pathways for (A) non-homologous end-joining (NHEJ) and (B) homology-directed repair (HDR). Specific proteins discussed in the article are featured. See text and ref.^,,,, for a more detailed discussion of these pathways.

Figure 2

Summary of approaches to increase HDR after CRISPR-Cas-induced DSBs Specific approaches discussed in the article are featured.

Figure 3

Schematic representation of cytosine and adenine base editors (A) Cytosine base editors (CBEs) are composed of a Cas9 nickase fused to a cytosine deaminase and one or two uracil glycosylase inhibitors (UGIs). CBEs convert C·G into T·A base pairs. (B) Adenine base editors (ABEs) are composed of a Cas9 nickase fused to a wild-type or mutant tRNA (tRNA) adenosine deaminase (e.g., TadA). ABEs convert A·T into G·C base pairs.

Figure 4

Schematic representation of a prime editor (PE) A PE consists of a fusion of Cas9 nickase and reverse transcriptase (RT). This complex is coupled with a prime editing guide RNA (pegRNA), which consists of the spacer, scaffold, reverse transcriptase template (RTT), primer binding sites (PBS), linker, and 3′ structural motifs (optional).

Figure 5

Schematic of Tn7-like RNA-guided transposon systems A Cascade/crRNA/TniQ complex is guided to the target site for insertion by the crRNA. TnsC and TnsA/B bind to the complex and increase the fidelity for the target site. Enzymatic integration of the donor DNA into the genome is mediated by TnsA/B.