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Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9   

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Original research article

A brief version of this protocol appeared in:
Science Translational Medicine
May 2016

Abstract

CRISPR-Cas9 based knockout strategies are increasingly used to analyze gene function. However, redundancies and overlapping functions in biological signaling pathways can call for generating multi-gene knockout cells, which remains a relatively laborious process. Here we detail the application of multi-color LentiCRISPR vectors to simultaneously generate single and multiple knockouts in human cells. We provide a complete protocol, including guide RNA design, LentiCRISPR cloning, viral production and transduction, as well as strategies for sorting and screening knockout cells. The validity of the process is demonstrated by the simultaneous deletion of up to four programmed cell death mediators in leukemic cell lines and patient-derived acute lymphoblastic leukemia xenografts, in which single cell cloning is not feasible. This protocol enables any lab with access to basic cellular biology equipment, a biosafety level 2 facility and fluorescence-activated cell sorting capabilities to generate single and multi-gene knockout cell lines or primary cells efficiently within one month.

Keywords: CRISPR, Cas9, Multiple gene knockout, Lentivirus, Primary human leukemia, Xenograft, sgRNA design

Background

Starting with curious initial observations of genetic elements known as clustered regularly interspaced short palindromic repeats (CRISPRs) within bacterial genomes (Ishino et al., 1987; Mojica et al., 2000) and subsequent gene editing in mammalian cells (Cong et al., 2013; Mali et al., 2013), CRISPR-Cas9 has become the cutting edge option for inexpensive and efficient gene editing. With successful application in cellular systems ranging from tobacco plant cells to zebrafish and primary human cells (Hsu et al., 2014), CRISPR-Cas9 can be directed by design of a short 20 nucleotide RNA sequence to create targeted DNA double strand breaks (DSB) within large genomes (Park et al., 2016). After DSBs occur, cells can initiate repair either through high fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ), often leading to small insertion and deletion (indel) mutations resulting in gene knockout (Gaj et al., 2013; Bétermier et al., 2014) (Figure 1).


Figure 1. Principle of genome editing by CRISPR Cas9. The principle of a gene knockout by CRISPR-Cas9 is shown exemplarily for the RIP1 sequence. A. Single guided RNA (sgRNA) consists of the target sequence specific crRNA (CRISPR RNA) and the constant tracrRNA (trans-activating crRNA) (Jinek et al., 2012). crRNA is binding to the genomic DNA adjacent to the PAM motif and tracrRNA guides the Cas9 enzyme to the locus. B. Cas9 mediated DNA double strand breaks (DSB) activate non-homologous end joining (NHEJ). C. Imprecise DSB repair leads to gain or loss of nucleotides (indels) with a two-thirds chance of causing frameshift mutations that may result in the generation of premature stop codons.

A number of different strategies have emerged to deliver Cas9 protein and targeting RNA into cells, including electroporation or transfection of Cas9/sgRNA ribonucleoprotein complexes, mRNA, plasmid or lentiviral vectors carrying sgRNA and Cas9 payloads (Sander and Joung, 2014; Shalem et al., 2014). Previously these LentiCRISPR plasmids carried a resistance gene to allow selection of cells with constitutive expression of the machinery necessary for CRISPR-Cas9-directed gene disruption.

As shown in our recent publication (McComb et al., 2016), we have adapted the LentiCRISPR protocol for directed disruption of several genes simultaneously in cell lines and primary leukemia cells based on selection by fluorescence combined with fluorescence-activated cell sorting (FACS). By swapping the puromycin resistance gene for fluorescent protein markers (EGFP, mCherry, tagBFP, or RFP657), up to four genes can be simultaneously targeted for CRISPR-Cas9-mediated gene disruption. Fluorescence-activated cell sorting enables isolation of cell lines or primary human cells bearing sgRNAs targeting one to four genes in one single experimental step. Our multi-color LentiCRISPR technique thus allows the simultaneous generation of knockout cells bearing anywhere between one and four gene knockouts, allowing rapid testing of gene-gene interaction within a set of genes of interest. The backbone vectors with the four different fluorescence markers as well as the herein described target constructs including cloning information have been deposited at Addgene.

Here we provide a complete step-by-step guide protocol to generate single and multi-gene knockout cells by multicolor LentiCRISPR (see Figure 2 for schematic overview).


Figure 2. Schematic overview of the procedure to generate multicolor LentiCRISPR knockout cell lines and patient derived xenografts

Development of the protocol
To study the regulation of cell death in leukemia, we developed multi-color LentiCRISPR as a tool to target proteins essential for two divergent pathways of programmed cell death, apoptosis and necroptosis (McComb et al., 2016). Efficient deletion of the respective targets, like RIP1, RIP3, MLKL, FADD and CASP8, was demonstrated by Western blot analysis of protein in targeted compartments (see Data analysis). Through simultaneous gene disruption, we showed that it is necessary to inactivate both apoptosis and necroptosis within leukemic cell lines and patient-derived xenografts in order to render cells resistant to SMAC mimetics, a specific class of chemotherapeutic compounds targeting the inhibitor of apoptosis proteins, IAPs. These data provide convincing evidence that both apoptosis and necroptosis can independently kill leukemia cells in vivo, and are a strong proof of concept for the multicolor LentiCRISPR technique as a means to investigate gene redundancy.

Experimental design
sgRNA design and preparation. We first describe a fast and easy way to design and clone sgRNAs for any target gene with a single pot reaction for restriction and ligation (Figure 3). We have had good success utilizing the CRISPR design online tool (http://cripr.mit.edu) from the Massachusetts Institute of Technology, developed by the lab of Feng Zhang to predict binding sites for Cas9 with minimal risk of off-target activity (Hsu et al., 2013). Alternative sgRNA prediction software (such as http://crispor.tefor.net/) can also be used to provide in silico prediction of sgRNA-specific cleavage activity based on a number of different algorithms. However, we still recommend the design of three sgRNAs per target gene and assessing their gene knockout activity in cell lines before moving on to more challenging applications. Strategies targeting only the 5’ exon of candidate genes might induce in-frame mutations that can retain the full protein functionality. A recent publication showed that targeting particular exonic regions with key functional protein domains increases the chance of null mutations without a full protein knockout (Shi et al., 2015). For this reason, we suggest spreading sgRNA candidates among different exons to increase the probability of achieving a potent gene deletion.


Figure 3. Principle of primer design and cloning for LentiCRISPR-Cas9 mediated gene knockout. Primer design is shown exemplarily for RIP1. A. From the genomic sequence the target locus for CRISPR editing was chosen and screened by http://crispr.mit.edu for sgRNA binding sites. B. After choosing a guide by score and location, complement primer sequence can be generated. C and D. For ligation into the Esp3I restriction site of the pLentiCRISPR plasmid sticky ends (CACC/CAAA) and the U6 transcriptional start site (TSS), (G) has to be added to the oligonucleotide sequence. U6, RNA Pol III promoter; EFS, EF1 short promoter.

Lentiviral production and infection. This protocol makes use of multicolor LentiCRISPR plasmids cloned from one-vector LentiCRISPR system developed by the Zhang lab (Shalem et al., 2014). Protocol conditions have been optimized for the transduction of acute lymphoblastic leukemia (ALL) cell lines and patient derived xenograft ALL cells from previously published protocols (Tiscornia et al., 2006; Kutner et al., 2009; Weber et al., 2012). Optimized conditions are recommended for every cell line or patient-derived sample. Here, we describe the production of lentiviral particles with the VSV-G envelope, because it is known for its high titers and broad tropism. Depending on the target cells the pseudotyping can be exchanged. For murine applications, the exchange to a mouse ecotropic envelope protein enhances safety and enables the use of the lentiviral vectors under biosafety level 1 conditions.

Analysis of knockout efficiency
After purification of cells transduced with Cas9/sgRNA targeted against a gene of interest, it is straightforward to confirm knockout at protein level (measurement via flow cytometry, ELISA or Western blot). Thus it is essential that a specific antibody for your protein of interest is available (see Figure 4 for further discussion and considerations for single cell cloning). Regardless of the viral transduction efficiency and the gene-editing efficacy of the selected sgRNA/CRISPR-Cas9 construct, DNA mutations do not invariantly lead to a loss of protein. Based on the triplet coding sequences the ratio of a frameshift after NHEJ is 2:1 resulting in possible indels without frameshift. Depending on the location of indel formation, this may also lead to unpredictable effects on mRNA or protein stability. Thus, we do not recommend knockout confirmation at DNA or RNA level by sequencing or SURVEYOR nuclease assay since this does not confirm the loss of the protein expression and function. Lesions can lead to a loss of amino acids and a change in protein functionality, but can also result in a slightly impaired protein with a near wild type function. Depending on the target gene a functional assay can be performed (i.e., enzyme activity) or cellular localization can be visualized (i.e., for nuclear receptors) but we recommend performing Western blots or ELISA to quantify protein level.

Limitations of the technique
Lentiviral vector efficiently delivers the CRISPR machinery to a wide range of cell lines and primary cells. However, as with any viral approach, there is a risk of insertional mutagenesis, although this can be minimized by transducing cells at a low multiplicity of infection (MOI) to limit the number of integration events. Constitutive expression of Cas9/sgRNA may also lead to accumulation of mutations at off-target sites, stressing the need for good sgRNA design to limit off-target binding. 

Efficiency of knockouts using LentiCRISPR can vary significantly depending on the specific genes targeted, especially for targets that confer a selective advantage/disadvantage to knockout cells. It might thus be a benefit to utilize an inducible CRISPR plasmid for such applications. In this case, Cas9 or sgRNA expression would be controllable in vitro and in vivo by administration of i.e., doxycycline. 
  Our strategy also depends on the reliability of the detection of reporter fluorescence. There is some evidence for silencing of expression over time for fluorescent proteins of the GFP family when expressed from lentiviral vectors. The populations should therefore be continuously monitored for expression. While lentiviral delivery has been proven to be very efficient in many different cellular systems, transduction efficiency heavily depends on the size of the delivered plasmids (Canté-Barrett et al., 2016). The large size of the LentiCRISPR vector is known to lead to low viral titers, nonetheless, viral concentration and other optimizations in the protocol below have allowed us to successfully apply these vectors in hard to transduce leukemic cell lines and primary cells.


Figure 4. Considerations for single cell cloning. We recommend single cell cloning if a clonal cell population with a constant genetic background is desired for long term experimentation. Here we present a Western blot confirmation of the knockout of RIP1 in either (A) double sorted or (B) single cell cloned Jurkat cells. Irrespective the purity of the sorted cell population, a minor RIP1 signal remains in the double sorted population, whereas in single cell clones a pure knockout can be achieved. Proteins were detected with mouse anti-RIP1 (1:1,000) and goat anti-mouse-HRP (1:5,000).

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Behrmann, L., McComb, S., Aguadé-Gorgorió, J., Huang, Y., Hermann, M., Pelczar, P., Aguzzi, A., Bourquin, J. and Bornhauser, B. C. (2017). Efficient Generation of Multi-gene Knockout Cell Lines and Patient-derived Xenografts Using Multi-colored Lenti-CRISPR-Cas9. Bio-protocol 7(7): e2222. DOI: 10.21769/BioProtoc.2222.
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