Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates

Abstract

Site-specific chemical conjugation of proteins can enhance their therapeutic and diagnostic utility but has seldom been applied to CRISPR-Cas9, which is a rapidly growing field with great therapeutic potential. The low efficiency of homology-directed repair remains a major hurdle in CRISPR-Cas9–mediated precise genome editing, which is limited by low concentration of donor DNA template at the cleavage site. In this study, we have developed methodology to site-specifically conjugate oligonucleotides to recombinant Cas9 protein containing a genetically encoded noncanonical amino acid with orthogonal chemical reactivity. The Cas9-oligonucleotide conjugates recruited an unmodified donor DNA template to the target site through base pairing, markedly increasing homology-directed repair efficiency in both human cell culture and mouse zygotes. These chemically modified Cas9 mutants provide an additional tool, one that is complementary to chemically modified nucleic acids, for improving the utility of CRISPR-Cas9–based genome-editing systems.

Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube

“…With regard to the former, our detection method of biolistic delivery in pollen and pollinated pistils would contribute to increasing the efficiency at various reproductive steps. In the latter regard, a number of approaches aimed at enhancing the efficiency of genome editing have been reported, including the modification of Cas9 (Ling et al 2020; Osakabe et al 2020), design of guide RNA (Moon et al 2019), the use of RNPs (Liang et al 2018; Svitashev et al 2016), and the use of small chemical compounds (Yu et al 2015). Various selection methods have also been reported, including those based on antibiotic resistance (Chesnokov and Manteuffel 2000) and herbicide resistance, by targeting endogenous genes (Han and Kim 2019).…”

CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future

“…This strategy was shown to facilitate HDR events while mitigating undesired NHEJ edits in human immortalized and stem cells (84,85). A more recent strategy combined a chemically modified Cas9 to the ssODN donor or a DNA adaptor that recruits the donor template, either of which improved HDR efficiency by localizing the donor template near the cleavage site (86). Despite these advancements, HDR is still achieved at a relatively low efficiency in eukaryotic cells and use of relatively harmful agents in cells such as NHEJ chemical inhibitors may not be ideal in a clinical setting.…”

Advances and Obstacles in Homology-Mediated Gene Editing of Hematopoietic Stem Cells

“…Furthermore, non-chemically modified ssODNs can be used. The authors report a 10-fold increase in HDR in HEK293 cells and a three-fold increase in mouse zygotes [ 117 ]. While these strategies have yet to be tested in HSPCs for diseases such as sickle cell, where large gene insertions are not required, this may prove to be beneficial.…”