How can I increase the frequencies of edited clones with only one modified allele and the other one encoding the wild-type sequence?

One of the most common genome editing outcomes is the introduction of an SNP in one allele and an indel in the other, such that it is especially challenging to obtain clones in which only one allele is modified (encoding for the desired edit) and the other one is unedited. To more efficiently obtain clones with SNP/wild-type heterozygosity, researchers have developed two strategies (Paquet et [...]

What are additional methods for improving HDR efficiency?

The development of novel methods for improving HDR efficiency is currently a major focus area in the genome editing field. Toward this end, the following approaches have shown promise: -Fusion or attachment of Cas9-sgRNA RNP and template (Aird et al. 2018; Savic et al. 2018; Carlson-Stevermer et al. 2017) -Restriction of Cas9 expression to S and G2 phases of the cell cycle, when the propensity [...]

What are the effects of chromatin configuration on HDR efficiency?

It has been demonstrated that nucleosomes inhibit target cleavage by SpCas9 in vivo, suggesting that Cas9 activity is influenced by chromatin structure in addition to the inherent activity of a given sgRNA (Yarrington et al. 2018). Although DNA sequence is a significant determinant of site-specific indel profiles, it has been shown that packaging of DNA into chromatin may influence editing [...]

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). [...]

Is it helpful to use HDR templates that introduce silent mutations in the PAM sequence or sgRNA seeding region?

Cas9-sgRNA RNPs can re-cut a given genomic target following HDR if the editing outcome does not involve alteration of the PAM or sgRNA seeding region in the protospacer sequence, such that the RNP complex can still bind to the genomic target. Given this, a common strategy for increasing the percentage of successful HDR in coding regions is the incorporation of silent mutations in HDR templates [...]