Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Summary

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

Abstract

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.

Introduction

The past few decades have seen numerous research efforts focused on identifying the contribution of key molecular pathways in acute myeloid leukemia (AML) pathogenesis. Traditionally, gene-disruption in AML cells has been performed using conditional knockout mice or short-hairpin RNA (shRNA). While knockout mice offer a sophisticated system for spatio-temporal control of gene-deletion, generating gene knockout mice is labor-intensive, time-consuming and expensive. Furthermore, gene-knockouts using recombination strategies is not easily scalable; these strategies do not lend themselves well to the interrogation of several genes in parallel. After the discovery of RNA i....

Protocol

1. Generating AML Cell Line Clones with High Expression of Stable and Active Cas9

1. Production of Cas9 lentivirus
   1. Day 0: Plate 4 x 10⁶ 293T cells in 10 mL of DMEM with 10% fetal bovine serum (FBS) and penicillin and L-glutamine in a 10 cm tissue culture dish in a biosafety Level 2 (BSL2) certified cell culture hood. Place the dish in a 37 °C incubator.
   2. Day 1: The plated 293T cells should be 70–80% confluent on day 1. Perform the transfection using the following protocol in the afternoon.
   3. Warm the transfection medium, culture media and transfection reagent to room temperature. Thaw all t....

Results

In our study, we first transduced the MOLM13 human AML cell line that bears the MLL-AF9 translocation with high-titer virus encoding the Cas9-blasticidin lentiviral plasmid. In our hands, bulk unsorted MOLM13-Cas9 cells did not display high level Cas9 expression by Western blotting and also did not perform well when assayed for efficient gene editing-using the method described previously⁷. Therefore, we proceeded to establish single cell clones and only select the .......

Discussion

In this manuscript, we describe a detailed protocol for conducting a CRISPR-Cas9-based competitive growth assay to investigate the role of candidate genes in AML cell lines using flow-cytometry in human/murine AML cells (Figure 5). The goal of the assay is to identify the effect of gene deletion on maintenance of AML cell proliferation over two to three weeks on a medium-throughput scale. Some critical steps need to be followed carefully to facilitate the scaling up of the described protocol.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
FLAG-M2 Antibody	sigma-aldrich	F3165, lot # SLBS3530V
Anti-mouse Antibody	Invitrogen	31446, lot # TA2514341
SuperSignal West Femto Maximum Sensitivity Substrate	Thermo Fisher	34095
ChemiDoc Imaging System	BIO RAD	17001401
Sorvall Legend RT centrifuge	Thermo Scientific
Blasticidin	Thermo Fisher	R21001
SYTOX Red	Thermo Fisher	S34859
Opti-MEM	Thermo Fisher	31985062
DMEM	Thermo Fisher	11965-092
RPMI	Thermo Fisher	11875-093
Penicillin-Streptomycin	Thermo Fisher	15140122
L-Glutamine (200 mM)	Thermo Fisher	25030081
Fetal Bovine Serum (FBS)	SAFC	12303C
single gRNA vector	Addgene #67974	pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W
CelLytic Nuclear extraction kit	sigma-alorich	NXTRACT
XtremeGENE 9	sigma-alorich	6365787001
Retronectin	Takara	T100B
Flow cytometer	BD Biosciences
T4 PNK	NEBioLabs	M0201S
T4 DNA ligation buffer	NEBioLabs	B0202S
T4 DNA Ligase enzyme	NEBioLabs	M0202S
Ampicillin	Fisher scientific	BP1760-25
LB agar	Fisher scientific	BP9724-500
LB Broth	Fisher scientific	BP9731-500
Qiagen mini-prep kit	Qiagen	27104
NanoDrop Spectrophotometer	Thermo Fisher	NanoDrop One
Recombinant Murine IL-3	Peprotech	213-13
Recombinant Murine IL-6	Peprotech	216-16
Recombinant Murine M-CSF	Peprotech	315-02
Stable competent cells	NEBiolabs	C3040I
10 cm Tissue Culture dishes	Fisher Scientific	353003
Cell lysis solution	Qiagen	158906
Protein precipitation solution	Qiagen	158910
DNA hydration solution	Qiagen	158914
QIAquick Gel Extraction Kit	Qiagen	28704
BbSI	New England BioLabs	R0539S
APEX 2.0x Taq Red Master Mix Kit	Genessee Scientific	42-138
Puromycin	Fisher scientific	BP2956100
50 mL polypropylene conical tubes	Fisher scientific	1495949A
15 mL polypropylene conical tubes	Fisher scientific	1495970C