How can I increase the frequencies of edited clones with only one modified allele and the other one encoding the wild-type sequence?
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 al. 2016):
-Use an sgRNA with a cut site farther away from the insertion site (>10 nt)
-Use an equal mix of mutant and wild-type HDR templates (one encoding for the SNP, the other encoding for the wild-type allele, both with the PAM sequence modified as explained in the previous FAQ)
Reference
Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 (2016).