Figure 2.

Screening strategies for indels, deletions, and small knock-in (KI). Screening to identify the CRISPR-edited hPSC clones harboring the desired indels, deletions, or small KI can be divided in three steps. The first step is a PCR-based screen to quickly detect clones with the on-target modification(s). For indels, the mutated clones can be identified by PCR followed by high resolution melt analysis (HRMA). To identify clones carrying a deletion, PCR with primers located at each side of the expected deleted sequence can be used. For clones carrying a small KI, if a restriction site has been added to the donor DNA template, enzymatic digestion of the PCR product that includes the targeted sequence will allow the detection of clones with on-target integration of the donor. Positive clones are then checked by Sanger sequencing to determine their exact sequence. This step is important to select clones with the desired modification(s) and to discard clones with unwanted events (i.e., in-frame events for KO projects, imperfect HDR events for a small KI project). Finally, in clones homozygous for the desired mutation, the transgene copy number is evaluated to ensure ploidy, whereas heterozygous and compound heterozygous clones are sub-cloned to determine the exact allelic sequences. PM, point mutation; WT, wild type; Het, heterozygous; Hom, homozygous; seq, sequencing; nt, nucleotides.