Human cell line HEK293T stably expressing PKC-Luc Human cell line HEK293T stably expressing NFAT-Luc Human cell line HEK293T stably expressing TCF/LEF-Luc Human cell line HEK293T stably expressing NEG-Luc

Introduction

The CRISPR-Cas9 system for genome editing is rapidly transforming research in biology and medicine, due to its ease-of-design, efficiency, specificity, and relatively low cost (Wang, et al., 2016). In mammalian cell culture systems, most genome editing is achieved using transient transfection or lentiviral transduction, which works well for routine, low-throughput applications. However, for other applications, it would be beneficial to have a system in which one component, namely the CRISPR-Cas9 nuclease or one of its derivatives, was stably integrated into the genome. In this Technical Note, we introduce GeneCopoeia’s GeneHero™ Cas9 stable cell lines, and discuss the great utility that these cell lines provide for genome editing applications.

CRISPR-Cas9 has been adapted from anti-viral immunity mechanisms present in many bacterial and archaea species. The most commonly used CRISPR-Cas9 system to date is from Streptococcus pyogenes (SpCas9). SpCas9 is a DNA endonuclease that, in the presence of a chimeric single guide RNA (sgRNA), makes a double strand break (DSB) in chromosomal DNA (Jinek, et al. 2012). Creation of a DSB is the necessary initiation event for most CRISPR-mediated genome editing applications

Why use a Cas9-expressing stable cell line?

In cell line-based genome editing, Cas9 and sgRNA are typically introduced into cells using transient transfection of either DNA or RNA, or Cas9 protein:sgRNA ribonucleoprotein (RNP) complexes. Frequencies of DSB formation, as measured in insertion/deletion (indel) assays, often exceed 60% (Fu, et al., 2013). Therefore, transfection of cell lines that do not have Cas9 previously integrated is usually sufficient for gene knockouts resulting from nonhomologous end joining (NHEJ), and for applications requiring donor-mediated, homology-directed repair (HDR), including gene knockout, gene mutagenesis, gene tagging, and Safe Harbor transgene knock-in.

There are additional applications that, while it is not required, are enhanced by having Cas9 stably integrated into the genome: 1) gene knockout using lentiviral CRISPR; 2) sgRNA library screening; 3) sgRNA functional validation; and 4) inducible gene editing. Each of these applications is discussed below.

Application 1: Gene knockout using lentiviral CRISPR

While transfection is commonly used for cell line-based genome editing, some cell lines do not transfect well using chemical-, lipid-, or electroporation-based approaches. For these cell lines, researchers often turn to lentiviral-mediated CRISPR CRISPR delivery, because lentiviruses efficiently infect a very large number of mammalian cell types. It is possible to express both Cas9 nuclease and an sgRNA from the same lentivirus, as has been shown previously (Sanjana, et al., 2014), or to co-transduce two different lentiviruses, one expressing Cas9 and the other expressing sgRNA. However, the titers of such “all in one” or Cas9-only lentiviral CRISPR viruses tend to be significantly lower than that achieved from sgRNA-only lentiviral vectors due to the large (>4.4 kb) size of the Cas9 gene. It is for this reason that GeneCopoeia recommends, for genome editing using lentiviral CRISPR applications, applications, that scientists transduce a Cas9-expressing stable cell line with sgRNA-only lentiviruses.

GeneCopoeia provides two options for lentiviral-based CRISPR-Cas9 tools. In the first option (the “DIY”, or do-it-yourself option), you can purchase lentiviral-based sgRNA-expressing plasmids using our online gene search tool, or by requesting a quote. GeneCopoeia will design and construct the plasmids for you. Once you receive your plasmids, you can package them into lentivirus particles yourself using GeneCopoeia’s Lenti-Pac™ packaging kits.

In the second option, GeneCopoeia will package your sgRNA lentiviral plasmids into particles for you. These particles are high titer (>108 TU/ml) and ready-to-use. More information is available on the GeneCopoeia lentiviral particle website. In any case, whether you are producing your own lentivirus, or having GeneCopoeia make it for you, you would use your lentiviral particles to infect a cell line stably expressing Cas9, then screen for your genetic modification and phenotype.

Application 2: sgRNA library screening

One of the most important applications for Cas9-expressing stable cell lines is sgRNA library screening. CRISPR sgRNA libraries are collections of hundreds-to-thousands of plasmids, each expressing a unique sgRNA, and are highly useful for forward mutagenesis screens (Sanjana, et al, 2014). GeneCopoeia carries pre-made CRISPR sgRNA libraries that cover several well-studied pathways and gene groups, and also offers custom sgRNA library construction. For more information, please visit our CRISPR sgRNA library page.

CRISPR sgRNA libraries enable researchers to knock out many genes simultaneously in mammalian cells, opening the door to high-throughput drug target identification and validation, phenotypic changes, and reporter assays. Previously, these applications were achieved using short hairpin RNA (shRNA)-based RNA interference (RNAi). However, while shRNA libraries are useful for high-throughput loss-of-function screens, RNAi has several disadvantages compared with CRISPR: 1) RNAi causes knockdown of gene expression, not knockout, so false negatives resulting from residual gene expression can be missed; 2) RNAi acts only on cytoplasmic RNA, and so cannot silence nuclear RNAs like long non-coding RNAs. Conversely, CRISPR makes permanent changes to the genetic code. Thus, CRISPR can make a complete knockout of all alleles of a gene, whether their transcription products are localized to the nucleus or cytoplasm. So, CRISPR sgRNA library screening can be straightforward, provided you have a good assay or phenotype for screening (Figure 1).

 

Figure 1. Workflow for CRISPR sgRNA libraries. A. Pooled screen. Cells infected with each sgRNA library pool are screened for the desired readout. Pooled cells are subjected to Sanger sequencing for individual sgRNAs, or deep sequencing to look for over- or under-representation of individual sgRNAs. B. GeneCopoeia sgRNA libraries are available either as pools or individual sgRNAs. Cells are infected with individual sgRNA lentiviruses. Wells are screened for the readout of interest. Individual sgRNAs corresponding to the phenotype of interest are already known without sequencing.

CRISPR sgRNA libraries are best suited for lentiviral delivery rather than plasmid transfection, because lentiviruses provide higher delivery efficiency. However, because the titer of Cas9-expressing lentiviruses is lower than sgRNA-expressing lentiviruses, most sgRNA libraries, including GeneCopoeia sgRNA libraries, are built without Cas9 in the vector. We recommend using our sgRNA libraries in Cas9-expressing stable cell lines.

One example of how useful GeneCopoeia’s Cas9-expressing stable cell lines are for sgRNA library screening comes from a paper published by Prashant Mali’s group at the University of California at San Diego (Shen et al., 2017), who used GeneCopoeia Cas9 stable cell lines with combinatorial sgRNA libraries to identify synthetic interactions among 73 cancer-associated genes. Most of these interactions were subsequently validated by drug treatments.

After their initial study, Mali’s group went on further to use sgRNA libraries in GeneCopoeia Cas9-expressing HeLa and A549 cell lines to knock out 51 genes encoding oxidative phosphorylation enzymes singly and in pairwise combinations (Zhao, et al. 2018). The researchers found, among both cell lines, 25 novel cases in which a single gene knockout had little to no effect on cell growth, whereas a pair of genes caused a significant growth defect. In addition, investigators observed that some single gene knockouts had a more severe growth phenotype in HeLa cells than in A549 cells. This difference in phenotype was due to the revelation that A549 cells, and not HeLa cells, carry a mutation in KEAP1, which encodes an E3 ubiquitin ligase that is mutated in many cases of lung cancer and has been implicated in resistance to chemotherapy. Loss of KEAP1 causes an elevation in the levels of some enzymes involved in oxidative phosphorylation, resulting in resistance to single oxidative phosphorylation gene knockout. The authors concluded that design of anticancer drugs targeting oxidative phosphorylation enzymes needs to be done in consideration of the KEAP1 mutational status of the cancer.

Application 3: sgRNA functional validation

Cell line-based CRISPR genome editing projects typically require several (>3) months of work, including transfection, clone isolation, and screening. While CRISPR typically provides a highly efficient method for genome editing, the individual sgRNAs themselves do exhibit variations in efficiency due to the nature of individual target sites. Therefore, it would be helpful to determine which CRISPR sgRNAs have the highest potential for successful genome editing before doing lots of screening work.

We recommend the mismatch cleavage assay (Qiu, et al., 2004) for sgRNA functional validation in a Cas9 stable cell line. GeneCopoeia’s IndelCheck™ mismatch cleavage assay (https://www.genecopoeia.com/product/indelcheck-detection-system/) is a cell culture-based assay designed to efficiently detect indels resulting from DSB-mediated NHEJ. The basic workflow for the IndelCheck™ mismatch cleavage assay for sgRNA functional validation is illustrated in Figure 2.

Figure 2. Workflow of the IndelCheck™ mismatch cleavage assay. A population of cells that has been exposed to CRISPR components is subjected to genomic DNA PCR using primers flanking the target site. Denaturation and reannealing of the PCR products creates a mixed population of homoduplex and heteroduplex molecules. The fragments are treated with T7 Endonuclease I, which cuts only the heteroduplex molecules. Cleavage products are detected by standard agarose gel electrophoresis. Functional validation of CRISPR sgRNAs can be performed either in a commonly used, “model” cell line (e.g., HEK293), or in your own cell line. GeneCopoeia has several pre-made human and mouse Cas9-expressing stable cell lines in widely-used backgrounds (Table 1). Alternatively, you can purchase CRISPR reagents from us that allow you to readily build your own Cas9-expressing stable cell line.

Application 4: Inducible genome editing

CRISPR is commonly used to generate genome modifications in a constitutive fashion, without regard to a specific time or stage in cell growth. Sometimes, though, it is useful to introduce the modification after a particular event has occurred, such as treatment of cells with a compound, or at a specific time during differentiation. Inducible CRISPR (iCRISPR) was first demonstrated by Gonzalez et al. (2014), who built an induced pluripotent stem cell (iPSC) model cell line carrying Cas9 that can be expressed after addition of the tetracycline analog doxycycline.