Clonally Selected Lines After CRISPR-Cas Editing Are Not Isogenic

Abstract

The CRISPR-Cas9 system has enabled researchers to precisely modify/edit the sequence of a genome. A typical editing experiment consists of two steps: (1) editing cultured cells; (2) cell cloning and selection of clones with and without intended edit, presumed to be isogenic. The application of CRISPR-Cas9 system may result in off-target edits, whereas cloning will reveal culture-acquired mutations. We analyzed the extent of the former and the latter by whole genome sequencing in three experiments involving separate genomic loci and conducted by three independent laboratories. In all experiments we hardly found any off-target edits, whereas detecting hundreds to thousands of single nucleotide mutations unique to each clone after relatively short culture of 10-20 passages. Notably, clones also differed in copy number alterations (CNAs) that were several kb to several mb in size and represented the largest source of genomic divergence among clones. We suggest that screening of clones for mutations and CNAs acquired in culture is a necessary step to allow correct interpretation of DNA editing experiments. Furthermore, since culture associated mutations are inevitable, we propose that experiments involving derivation of clonal lines should compare a mix of multiple unedited lines and a mix of multiple edited lines.

FIG. 1.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9) edited clones are not isogenic. (A) In three different experiments conducted by three different groups, CRISPR-Cas9 unedited (in blue circles) and edited (in black circles) single cell clones were analyzed. Orange-filled circles represent clones with CNA. (B) Copy number profiles for the analyzed samples. CNA inherited from unedited parental lines are shown between orange broken lines. In the experiment conducted at Yale, the heterozygous deletion on chromosome 20 was mosaic in the parental unedited iPSC line (90% of cells) [see (A)], and two derived clones (unedited and edited) inherited it. Similarly, in the experiment done at Mayo, multiple mosaic CNA in the parental line (such as duplication on chromosome 1 and deletion of chromosome 14) were inherited by only one out of three clones. In addition, one edited line, CP-601-40, was genomically unstable. In the experiment by OMRF/BCM, a mosaic duplication on chromosome 20 inherited from the unedited parental iPSC line was present in only one of the clones. Clones were selected to avoid mosaic duplication on chromosome 1 present in the parental line. (C) Distribution of private SNVs by allele frequency in each clone (Supplementary Table S1). Clones have hundreds and thousands of point mutations that arose before cloning during culturing of parental lines (see Supplementary Fig. S5 for variant calling). OMRF, Oklahoma Medical Research Foundation; BCM, Baylor College of Medicine; CNA, copy number alteration; iPSC, induced pluripotent stem cell; SNV, single nucleotide variation.