DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing

Abstract

Many clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based genome editing technologies take advantage of Cas nucleases to induce DNA double-strand breaks (DSBs) at desired locations within a genome. Further processing of the DSBs by the cellular DSB repair machinery is then necessary to introduce desired mutations, sequence insertions, or gene deletions. Thus, the accuracy and efficiency of genome editing are influenced by the cellular DSB repair pathways. DSBs are themselves highly genotoxic lesions and as such cells have evolved multiple mechanisms for their repair. These repair pathways include homologous recombination (HR), classical nonhomologous end joining (cNHEJ), microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA). In this review, we briefly highlight CRISPR-Cas9 and then describe the mechanisms of DSB repair. Finally, we summarize recent findings of factors that can influence the choice of DNA repair pathway in response to Cas9-induced DSBs.

Figure 1.. CRISPR-Cas9 delivery.

(A) Mechanism of CRISPR-Cas9 genome editing. A sgRNA/tracrRNA-crRNA associates with the Cas9 endonuclease to form the Cas9-gRNA complex. The gRNA guides Cas9 to its target site of the genomic DNA by recognizing the protospacer-adjacent motif (PAM). When Cas9 binds to the correct dsDNA target, its HNH and RuvC nuclease domains cleave the DNA to yield either blunt or staggered ends. Genome editing is achieved during the repair of the resulting DSB, resulting in precise mutations, gene deletions, or sequence insertions. (B) Cas9 can be delivered in three forms. First, introducing Cas9-gRNA ribonucleoprotein (RNP) complex and DNA templates directly through nanoparticles, electroporation, or microinjection. Second, delivering a plasmid DNA or viral vector for Cas9 and gRNA production in situ. Third, delivering separate gRNA together with mRNA for Cas9 protein expression inside the cell. With the exception of microinjection, during which Cas9 components can be directly injected into the nucleus, other delivery methods release Cas9 components into the cytosol. DNA sensing receptors in endosome or cytosol, including the Toll-like receptor 9 (TLR9), melanoma 2 (AIM2) and cyclic GFP-AMP synthase-STING (cGAS-STING), drive cell immune responses to foreign DNA. mRNA can also be recognized by TLR7 and TLR8 in endosome causing mRNA degradation and type I interferon α (IFN-α)-mediated immune response. Cas9 in the cytosol can be directed into nucleus through nuclear localization sequence; how plasmid DNA, viral DNA and DNA templates enter the nucleus is unknown.

Figure 2.. The four major pathways to repair DNA Double-Strand Breaks (DSBs).

(A) Unprocessed DSBs can be repaired through classic non-homologous end joining (cNHEJ) allowing the two ends of the DSB to be re-ligated. (B) DSB ends can also be processed by the MRN complex and its interacting factors to yield short 3’ ssDNA overhangs. (C) The short 3’ ssDNA overhangs can then be channeled into the microhomology-mediated end joining (MMEJ) pathway. (D) Alternatively, the DSB ends can undergo further long-range resection by either EXO1 or BLM/DNA2. These longer ssDNA overhangs are first bound by RPA and can then be channeled into the (E) SSA pathway, which is mediated by the protein RAD52. (F) Alternatively, the RPA-ssDNA can serve as a substrate for the RAD51 filament assembly, allowing the resulting DNA intermediates to be directed towards repair by (G) HR. For HR, both ssDNA and dsDNA templated homology repair (HDR) pathways are shown.

Figure 3.. Factors affecting DNA repair outcomes of Cas9-induced DSBs.

(A) DSB repair pathways during the cell cycle. cNHEJ is active throughout the cell cycle (black circle). MMEJ and HDR can only be employed in S/G2 phases (green circle). (B) In the absence of a template guided repair mechanism editing outcomes are significantly affected by the target site sequence. MMEJ efficiency and the pattern of DNA small deletions are dependent upon the presence of microhomologies within the first 10 bp from the DSB end. 1–2 bp insertions/deletions through cNHEJ is affected by the nucleotide at the 4^th position upstream from the PAM. (C) Nucleosomes can potentially block Cas9 access to target site, although the site can be exposed through nucleosome breathing or by a nucleosome remodeler. Compacted heterochromatin promotes HR, MMEJ, and SSA, while cNHEJ is preferred in euchromatin. (D) Homology-directed repair (HDR) with ssDNA or dsDNA donor templates. Double-stranded DNA donor templated repair (DSTR) occurs mainly through HR and single-stranded DNA donor templated repair (SSTR) occurs mainly through SSA and SDSA. Asymmetric HDR can arise when one end is repaired through HR, while the other end is repaired through cNHEJ or MMEJ. With ssDNA, the end that is complementary to the 3’ end of ssDNA templates is repaired through SSA/SDSA, while how the other end is repaired is unknown. The repair of the other end through MMEJ generates asymmetric HDR which displays a bias directionality with respect to the orientation of the ssDNA templates.

Figure 4.. The outcomes of Cas9-induced DNA double-strand break repair.

Once Cas9-gRNA complex binds to its target site, the HNH domain accurately cuts at the target strand −3 bp upstream of the PAM, while the RuvC-like domain cuts the −3, −4, or −5 positions of non-target strand. These cleavages can generate DSBs with either blunt ends or 1–2 nt staggered ends. Blunt ends can be ligated directly through cNHEJ without introducing any mutations, while staggered ends need to be filled or cleaved before ligation resulting 1–2 bp insertions or deletions. End resection directs DSB repair through template-independent MMEJ or template-dependent HDR. For MMEJ, small homologous DNA sequences (5–25 bp) within the two resected ends are paired and lead to DNA repair resulting in small deletions or insertions. In the presence of either ssDNA or dsDNA templates, homology directed repair (HDR) compete with cNHEJ and MMEJ for precise DNA repair.