Methodologies for Improving HDR Efficiency

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is a precise genome manipulating technology that can be programmed to induce double-strand break (DSB) in the genome wherever needed. After nuclease cleavage, DSBs can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathway. For producing targeted gene knock-in or other specific mutations, DSBs should be repaired by the HDR pathway. While NHEJ can cause various length insertions/deletion mutations (indels), which can lead the targeted gene to lose its function by shifting the open reading frame (ORF). Furthermore, HDR has low efficiency compared with the NHEJ pathway. In order to modify the gene precisely, numerous methods arose by inhibiting NHEJ or enhancing HDR, such as chemical modulation, synchronized expression, and overlapping homology arm. Here we focus on the efficiency and other considerations of these methodologies.

Keywords: CRISPR-Cas9; DSB; HDR; HDR enhancement; NHEJ; NHEJ inhibitors; cell arrest.

Figure 1

Canonical non-homologous end joining (c-NHEJ). After CRISPR-Cas9 introduced a DSB, NHEJ is initiated by the binding of the Ku heterodimeric complex. This then forms the core complex, which is considered to recognize broken ends and keeps them together. The ends will then be ligated by various ligases.

Figure 2

Homology-directed repair HDR. When DSB happens in the S- or G2-phase of the cell cycle and homologous sequence exists near the DSB, DSB can be handled through the HDR pathway if the ends of the DSB are resected. Ends will be coated with various proteins and then invade homologous duplex DNA to form an exchange intermediate: the D-loop structure. Most D-loop structures will be extended by DNA synthesis (dashed arrow). The second end pairs to the D-loop and starts extension. This pathway is called the double Holliday junction pathway. Ligation generates the characteristic double Holliday junction, which may be cleaved by HJ resolvases into either crossover or non-crossover products. The synthesis-dependent strand annealing pathway is illustrated on the right. After D-loop formation, replication and branch migration take place which can lead to D-loop translocation. The translocating D-loop is unstable and collapses easily. After collapse, the extended first end may anneal to complementary ssDNA in the resected second end. Replicative extension of both ends and ligation generates non-crossover products.