Fig. 2

Assay work flow summary for single cell CRISPR-Cas9 essentiality assay. Cells are infected with pLentiCRISPR containing Cas9 and a validated sgRNA targeting a gene of interest. After virus infection, the cells are selected with puromycin for a minimum of 2 weeks before single cell dilution in 384-well plates. Blue wells labeled with two “XX” indicate cells with homozygous disrupted alleles, orange with “X” indicate cells with heterozygous disrupted alleles, and yellow indicate cells in which the target alleles are unedited. Two medium-throughput methods are used to examine the zygosity of the gene of interest in each of the isolated clones. For the restriction digest, the region around the sgRNA target site is amplified by PCR and subject to restriction digest. PCR primers are chosen such that DNA fragments of unequal length are produced if the restriction site remains intact. Alleles that have been edited should have the restriction site disrupted (represented by the green band) and therefore will not be cut by the restriction enzyme. Unedited alleles (represented by the pink band) should retain an intact restriction site and should be cut by the restriction enzyme. The gel shows examples of clones in which both alleles have been edited (blue asterisk), one allele has been edited (orange asterisk) and no alleles have been edited (yellow asterisk). Fluorescent fragment length analyses are also used to assess indel status. PCR primers that incorporate a fluorescent tag are used to amplify an area around the sgRNA binding sequence and the Cas9 cut site (indicated by the pink band). A fragment of un-edited length suggests a lack of disruption, whereas a fragment that is either longer or shorter than the un-edited length suggests the presence of an indel (indicated by the green band)