title: A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality.
abstract: BACKGROUND
  Target selection for oncology is a crucial step in the successful development of therapeutics. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 editing of specific loci offers an alternative method to RNA interference and small molecule inhibitors for determining whether a cell line is dependent on a specific gene product for proliferation or survival. In our initial studies using CRISPR-Cas9 to verify the dependence on EZH2 activity for proliferation of a SMARCB1/SNF5/INI1 mutant malignant rhabdoid tumor (MRT) cell line, we noted that the initial reduction in proliferation was lost over time. We hypothesized that in the few cells that retain proliferative capacity, at least one allele of EZH2 remains functional. To verify this, we developed an assay to analyze 10s-100s of clonal cell populations for target gene disruption using restriction digest and fluorescent fragment length analyses.
  
  
  RESULTS
  Our results clearly show that in cell lines in which EZH2 is essential for proliferation, at least one potentially functional allele of EZH2 is retained in the clones that survive.
  
  
  CONCLUSIONS
  This assay clearly indicates whether or not a specific gene is essential for survival and/or proliferation in a given cell line. Such data can aid the development of more robust therapeutics by increasing confidence in target selection.
authors.affiliation: Epizyme, Inc., 400 Technology Square, 4th Floor, Cambridge, MA 02139 USA.
authors.forename: A R
authors.initials: AR
authors.lastname: Grassian
authors.affiliation: Horizon Discovery Ltd, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL UK.
authors.forename: T M E
authors.initials: TME
authors.lastname: Scales
authors.affiliation: Epizyme, Inc., 400 Technology Square, 4th Floor, Cambridge, MA 02139 USA.
authors.forename: S K
authors.initials: SK
authors.lastname: Knutson
authors.affiliation: Epizyme, Inc., 400 Technology Square, 4th Floor, Cambridge, MA 02139 USA.
authors.forename: K W
authors.initials: KW
authors.lastname: Kuntz
authors.affiliation: Horizon Discovery Ltd, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL UK.
authors.forename: N J
authors.initials: NJ
authors.lastname: McCarthy
authors.affiliation: Horizon Discovery Ltd, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL UK.
authors.forename: C E
authors.initials: CE
authors.lastname: Lowe
authors.affiliation: Horizon Discovery Ltd, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL UK.
authors.forename: J D
authors.initials: JD
authors.lastname: Moore
authors.affiliation: Epizyme, Inc., 400 Technology Square, 4th Floor, Cambridge, MA 02139 USA.
authors.forename: R A
authors.initials: RA
authors.lastname: Copeland
authors.affiliation: Epizyme, Inc., 400 Technology Square, 4th Floor, Cambridge, MA 02139 USA.
authors.forename: H
authors.initials: H
authors.lastname: Keilhack
authors.affiliation: Epizyme, Inc., 400 Technology Square, 4th Floor, Cambridge, MA 02139 USA.
authors.forename: J J
authors.initials: JJ
authors.lastname: Smith
authors.affiliation: Horizon Discovery Ltd, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL UK.
authors.forename: J A
authors.initials: JA
authors.lastname: Wickenden
authors.affiliation: Epizyme, Inc., 400 Technology Square, 4th Floor, Cambridge, MA 02139 USA.
authors.forename: S
authors.initials: S
authors.lastname: Ribich
keywords: CRISPR; EZH2; Epigenetics; Target identification; Target validation