Does the HDR insertion site need to be directly next to the PAM?
Does the HDR insertion site need to be directly next to the PAM?
It is highly recommended that the HDR insertion site is positioned within 10 nt upstream or downstream of the Cas9-sgRNA RNP cut site. For wild-type SpCas9, this is located 3–4 nucleotides upstream of the PAM. It has been demonstrated that there is an inverse relationship between HDR efficiency and the distance between the insertion site and Cas9-gRNA RNP cut site (Paquet et al. 2016).
References
Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 (2016).