Fig. 6. DEC can increase the HDR rate of Cas9 RNP through reversible donor DNA conjugation. (a) Overview of the workflow for conjugating the donor DNA to the Cas9 RNP. The Cas9 RNP is first conjugated to PEG–DEC–N₃ to generate Cas9 RNP DEC–N₃. Donor DNA with a DBCO handle is then clicked with the Cas9 RNP–DEC–N₃ and electroporated into cells. The donor DNA is released from the Cas9 RNP in the reducing environment of the cells, this conjugation strategy ensures that cells with the Cas9 RNP also have donor DNA. (b) Comparison of the HDR efficiency between physically mixed Cas9 RNP/donor DNA and Cas9 RNP–donor DNA conjugate (Cas9 RNP–DEC–DNA) after electroporation. Cas9 RNP–DEC–DNA had a 50% increase in hdr over cells treated with a physical mixture of Cas9 RNP and donor DNA.