Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing.

Due to post-cleavage residence of the Cas9-sgRNA complex at its target, Cas9-induced DNA double-strand breaks (DSBs) have to be exposed to engage DSB repair pathways. Target interaction of Cas9-sgRNA determines its target binding affinity and modulates its post-cleavage target residence duration and exposure of Cas9-induced DSBs. This exposure, via different mechanisms, may initiate variable DNA damage responses, influencing DSB repair pathway choices and contributing to mutational heterogeneity in genome editing. However, this regulation of DSB repair pathway choices is poorly understood. In repair of Cas9-induced DSBs, repair pathway choices vary widely at different target sites and classical nonhomologous end joining (c-NHEJ) is not even engaged at some sites. In mouse embryonic stem cells, weakening the target interaction of Cas9-sgRNA promotes bias towards c-NHEJ and increases target dissociation and reduces target residence of Cas9-sgRNAs in vitro. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ aggravates off-target activities of Cas9-sgRNA due to its weak interaction with off-target sites. By dislodging Cas9-sgRNA from its cleaved targets, DNA replication alters DSB end configurations and suppresses c-NHEJ in favor of other repair pathways, whereas transcription has little effect on c-NHEJ engagement. Dissociation of Cas9-sgRNA from its cleaved target by DNA replication may generate three-ended DSBs, resulting in palindromic fusion of sister chromatids, a potential source for CRISPR/Cas9-induced on-target chromosomal rearrangements. Target residence of Cas9-sgRNA modulates DSB repair pathway choices likely through varying dissociation of Cas9-sgRNA from cleaved DNA, thus widening on-target and off-target mutational spectra in CRISPR/Cas9 genome editing.

Liu SC ，Feng YL ，Sun XN ，Chen RD ，Liu Q ，Xiao JJ ，Zhang JN ，Huang ZC ，Xiang JF ，Chen GQ ，Yang Y ，Lou C ，Li HD ，Cai Z ，Xu SM ，Lin H ，Xie AY ... -  《GENOME BIOLOGY》

Target binding and residence: a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing.

Feng Y ，Liu S ，Chen R ，Xie A ... -  《-》

Methods Favoring Homology-Directed Repair Choice in Response to CRISPR/Cas9 Induced-Double Strand Breaks.

Yang H ，Ren S ，Yu S ，Pan H ，Li T ，Ge S ，Zhang J ，Xia N ... -  《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》

Proximal binding of dCas9 at a DNA double strand break stimulates homology-directed repair as a local inhibitor of classical non-homologous end joining.

Feng YL ，Liu SC ，Chen RD ，Sun XN ，Xiao JJ ，Xiang JF ，Xie AY ... -  《-》

Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in Leishmania.

CRISPR-Cas9 genome editing relies on an efficient double-strand DNA break (DSB) and repair. Contrary to mammalian cells, the protozoan parasite Leishmania lacks the most efficient nonhomologous end-joining pathway and uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair to repair DSBs. Here, we reveal that Leishmania predominantly uses single-strand annealing (SSA) (>90%) instead of MMEJ (<10%) for DSB repair (DSBR) following CRISPR targeting of the miltefosine transporter gene, resulting in 9-, 18-, 20-, and 29-kb sequence deletions and multiple gene codeletions. Strikingly, when targeting the Leishmania donovani LdBPK_241510 gene, SSA even occurred by using direct repeats 77 kb apart, resulting in the codeletion of 15 Leishmania genes, though with a reduced frequency. These data strongly indicate that DSBR is not efficient in Leishmania, which explains why more than half of DSBs led to cell death and why the CRISPR gene-targeting efficiency is low compared with that in other organisms. Since direct repeat sequences are widely distributed in the Leishmania genome, we predict that many DSBs created by CRISPR are repaired by SSA. It is also revealed that DNA polymerase theta is involved in both MMEJ and SSA in Leishmania Collectively, this study establishes that DSBR mechanisms and their competence in an organism play an important role in determining the outcome and efficacy of CRISPR gene targeting. These observations emphasize the use of donor DNA templates to improve gene editing specificity and efficiency in Leishmania In addition, we developed a novel Staphylococcus aureus Cas9 constitutive expression vector (pLdSaCN) for gene targeting in LeishmaniaIMPORTANCE Due to differences in double-strand DNA break (DSB) repair mechanisms, CRISPR-Cas9 gene editing efficiency can vary greatly in different organisms. In contrast to mammalian cells, the protozoan parasite Leishmania uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair (HDR) to repair DSBs but lacks the nonhomologous end-joining pathway. Here, we show that Leishmania predominantly uses single-strand annealing (SSA) instead of MMEJ for DSB repairs (DSBR), resulting in large deletions that can include multiple genes. This strongly indicates that the overall DSBR in Leishmania is inefficient and therefore can influence the outcome of CRISPR-Cas9 gene editing, highlighting the importance of using a donor DNA to improve gene editing fidelity and efficiency in Leishmania.

Zhang WW ，Matlashewski G 《mSphere》