However, it is preferable to design features into the genome editing strategy that facilitate identification of clones with the desired genotype without relying on Sanger sequencing. In cases where a simple deletion is desired, using two gRNAs spaced ~100 bp apart may slightly increase mutation efficiency10 and will facilitate genotyping of individual clones. For HDR, the DNA donor could be designed to insert or remove a restriction endonuclease site (Box 1). If these features are incorporated into the genome editing strategy, then they can be substituted for Steps 37–47 and 51–54.
