Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.
The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.
Stable genetic alterations provide an advantage over transient methods of cellular perturbation, which can be variable in their efficiency and duration. Genomic editing has become increasingly common in recent years due to the development of target specific nucleases, such as zinc-finger nucleases1,2,3,4,5, transcription activator-like effector nucleases (TALENs)6,7,8,9 and RNA-guided nucleases derived from the clustered, regularly interspaced short palindromic repeats (CRISPR) system10.
The CRISPR/Cas9 editing machinery is adapted from an immune system that bacteria and archaea use to defend against viral infections11,12,13. In this process, short, 20-30 nt fragments of invading viral sequence are incorporated into a genomic locus as "spacers" flanked by repeating units14,15. Subsequent transcription and RNA processing generates small CRISPR-associated RNAs16 (crRNAs) that, together with a trans-activating crRNA17 (tracrRNA), assemble with the effector Cas9 endonuclease. The crRNAs thus provide specificity to Cas9 targeting, guiding the complex to cleave complementary viral DNA sequences and preventing further infections18,19. Any "protospacer" sequence in the targeted DNA can serve as the source of the crRNA, as long as it is directly 5' to a short protospacer adjacent motif (PAM), NGG in the case of S. pyogenes Cas920. The absence of the PAM sequence near the spacer in the host's CRISPR locus distinguishes between self and non-self, preventing targeting of the host. Because of its universality and flexibility, this biological system has been powerfully adapted for genomic editing, such that nearly any PAM-adjacent DNA site can be targeted. In this version, a further modification fused the crRNA and tracrRNA into a single guide RNA (sgRNA) component that is loaded into the Cas9 protein21.
Upon expression of Cas9 and an sgRNA in eukaryotic cells, the Cas9 protein cleaves both DNA strands at the targeted locus. In the absence of a suitable region of homologous sequence, the cell fixes this break via non-homologous end joining (NHEJ)22,23,24, which typically introduces small deletions or, rarely, insertions. When targeting an open reading frame, the repair likely leads to a translational frameshift that produces a non-functional protein product. In contrast, when provided with an exogenous template with large regions of homology, the cell may fix the double-strand break by homology directed repair25,26. This route allows for larger precise deletions, replacements or insertions in the genome, coupled with the introduction of excisable selection markers27.
Here, we present protocols for generating knockout cell lines by either of these two CRISPR/Cas9 methods (Figure 1A). The NHEJ approach uses a single sgRNA cut site and selection-independent screening, and thus requires little upfront preparation. When using this method, guide RNAs complementary to exons near the 5' end of the transcript, which are most likely to produce a knockout, must be designed. Since the modifications to the genome in this case are small, screening for knockout clones is based on dot blots, where the protein product is assessed in a high-throughput manner. We use the generation of ELAV-like 1 protein (ELAVL1) knockout lines as an example. The second approach relies on homology directed repair (HDR) and uses two sgRNA cut sites that span the gene or region of interest, allowing for deletions of tens of kb. A plasmid with two regions of homology that flank the cleavage sites provides a replacement template (Figure 1B), introducing a selectable resistance marker that increases efficiency of knockout generation. This method can also be adapted to introduce gene modifications with properly designed homology arms. In this case, the integration of a new DNA fragment allows for PCR based screening (Figure 1C). Here, we use the generation of Pumilio RNA binding family member 2 (PUM2) knockout lines as an example.
1. Identification of Homology Regions Around the Desired Deletion
NOTE: Only necessary if using selection-based editing.
2. Generation of Cas9-sgRNA Expression Plasmids
3. Generation of Homology-directed Repair Template Plasmids
NOTE: Only necessary if using selection-based editing. The homology-directed repair template plasmid consists of a drug resistance cassette flanked by two regions that are complementary to the genome just outside the two sgRNA target sites (Figure 1B).
Right homology arm PCR product | 0.06 pmol (30-40ng) |
Left homology arm PCR product | 0.06 pmol (30-40ng) |
pGolden-Neo plasmid (100 ng/µL) | 1 µL |
pUC19-BsaI plasmid (100 ng/µL) | 1 µL |
2x T7 DNA ligase buffer | 5 µL |
BsaI (10 U/µL) | 0.75 µL |
T7 DNA ligase (3000 U/µL) | 0.25 µL |
Water | up to 10 µL |
Dilute colony template | 1.25 µL |
10x Taq reaction buffer | 1.25 µL |
20mM dNTPs | 0.25 µL |
10 µM Forward primer | 0.25 µL |
10 µM Reverse primer | 0.25 µL |
Taq Polymerase | 0.25 µL |
Water | 9 µL |
4. Transfection of CRISPR Components into Cultured Cells
5. Drug Selection
6. Isolation of Clonal Populations
7. Screening Candidates
Cell lysate | 0.5 µL |
10x KOD buffer | 1.25 µL |
25mM MgSO4 | 0.75 µL |
2 mM dNTPs | 1.25 µL |
10 µM Forward primer | 0.375 µL |
10 µM Reverse primer | 0.375 µL |
KOD polymerase | 0.25 µL |
Water | 7.75 µL |
8. Verify the Genomic Mutation by Sequencing
For the generation of ELAVL1 knockout lines, a robust antibody was available, so editing using single sgRNAs (Figure 1A, left) was performed, followed by dot immunoblot. Three sgRNAs were transfected independently to compare efficiencies and to rule out off target effects in the resulting clones. After collecting and blotting cell lysates from clonal populations onto two nitrocellulose membranes, the blots were probed for both ELAVL1 and PUM2 (as a normalization control) (Figure 2A). The quantified ratio of ELAVL1 to PUM2 signal demonstrated a very broad, >104-fold range among the isolated lines (Figure 2B). Using this ratio as a selection criterion, we validated a number of lines using ELAVL1 western blots (Figure 3A). In most cases, clonal populations with the lowest relative ELAVL1 signal were correctly identified as knockouts (Figure 2B), confirming the predictive power of this quantification (despite considerable variability in the control signal, which likely arose from differing numbers of collected cells). Conversely, most of the clones with intermediate ratios exhibited ELAVL1 signal upon validation, potentially representing monoallelic deletion events. However, no general tiering of the ratios into WT, monoallelic and full (biallelic) mutant pools was observed, and even the ELAVL1-positive clones displayed a wide and continuous range of levels, likely due to clonal variability of expression. The overall effectiveness of ELAVL1 knockout generation using this method ranged from 6 to 36% among the sgRNAs.
For other gene targets (such as PUM2), the quality of the available antibodies prevents accurate screening of candidate clones by dot blot due to high background from cross-reactivity. Additionally, the efficiency of targeting some genomic loci may be substantially lower due to chromatin accessibility. In such cases, a selection-dependent method is appropriate to increase efficiency. Two sgRNAs targeting early and late coding regions of PUM2 were cotransfected with a homology-directed repair template in order to excise 45 kb of genomic DNA and replace it with a neomycin resistance cassette (Figure 1A, right). Assembly of the homology template is illustrated in Figure 1B. PCR primers were designed to amplify regions around the homology arms based on either successful or unsuccessful integration of the resistance cassette (Figure 2C), and were tested on untransfected and the bulk selected cells (left panel). As expected, in untransfected cells, only an amplicon corresponding to the endogenous state (top) was observed, while the bulk selected cells showed amplicons from both wild-type and mutant alleles. Cell lysates from generated clonal candidates were collected and screened for the presence of both the endogenous sequence and the resistance cassette. Candidate knockouts were identified by the presence of the resistance cassette amplicon alone, indicating biallelic deletion/integration (Figure 2D, stars). Clones with monoallelic insertions were also observed, and were fairly common, since such populations can also survive the selection process. These clones were indicated by the presence of both the endogenous sequence and the resistance cassette. In a few cases, clonal wildtypes at the tested locus were also observed, suggesting that random, non-sgRNA-assisted genomic integration of the homology-directed repair template occurs at a low frequency. Biallelic mutant lines were further validated by western blot (Figure 3B). The overall efficiency of knockout generation using the HDR procedure was 33%.
Figure 1: A workflow for generating CRISPR-mediated knockouts in mammalian cell lines. (A) Genome editing events produced by CRISPR/Cas9 cleavage and NHEJ (left) or HDR (right). The site of Cas9 cleavage is denoted by a white vertical line. (B) Components and assembly of the HDR template plasmid. The type IIs restriction enzymes used in Golden Gate cloning, such as BsaI, recognize an asymmetric DNA sequence and leave a staggered cut outside of the recognition sequence, allowing for the design of arbitrary overlapping segments for assembly (pictured). Arrows indicate the primer positions to check for correct plasmid assembly. (C) Workflow: after transfection (and drug selection if applicable), cells are plated at limiting dilutions in 96-well plates. Clonal populations are screened either by dot immunoblot (selection-independent, NHEJ) or colony PCR (selection-dependent, HDR). Please click here to view a larger version of this figure.
Figure 2: Screening candidate knockout lines by dot blots or colony PCR. (A) Dot immunoblot of isolated ELAVL1-targeted clones with ELAVL1 antibody (left), and separately with an antibody for a control protein, Pum2 (right). Clones that displayed little control signal, likely due to low input protein amounts, are marked with an X and excluded from further analysis. Clones with low ELAVL1 signal (circles) were further tested by western blots that confirmed (green) or invalidated (grey) the candidates. (B) Quantification of KO candidates from dot blot. Ratios of ELAVL1 to control protein signal in the dot blot shown in A, in increasing order. (C) Primer design for knockout/insertion testing via colony PCR. A primer complementary to the genomic locus outside of the homology arm region (red) is paired with a primer complementary to the endogenous sequence (blue) or to the resistance cassette (green), probing for the unedited state, or a deletion/insertion event, respectively. Separate primer pairs are designed to test both sides of the integration. (D) Example agarose gel of colony PCR results using primers spanning the upstream integration junction using endogenous inside primers (top) and knockout inside primers (bottom). Primers are first validated in both parental T-REx293 cells and bulk selected cells (left). Individual candidate clones (middle) and the expected band patterns for WT, KO and monoallelic deletion (WT/KO) clones (right). Biallelic knockout clones are indicated by a star. The correct amplification products are indicated by black arrows. Please click here to view a larger version of this figure.
Figure 3: Validation of knockout candidates by Western blot. (A) Western blot for ELAVL1 (top) of a subset of clones identified by dot blot. PUM2 was blotted on the same membrane as a loading control (bottom). (B) Western blot for PUM2 (top) of a subset of clones identified by colony PCR. GAPDH was blotted on the same membrane as a loading control (bottom). Please click here to view a larger version of this figure.
The CRISPR/Cas9 system has allowed for efficient generation of stable genomic modifications, which provide a more consistent alternative to other transient manipulation methods. Here, we have presented two methods for rapid identification of CRISPR/Cas9 gene knockouts in mammalian cell lines. Both methods require little cellular material, so testing can be done in early stages of clonal culture, saving time and reagents. To increase the efficiency of both methods, we recommend testing multiple sgRNAs, as efficiencies vary depending on sequence and genomic location. In addition, using multiple sgRNAs is useful in identifying off-target effects by detecting outlier phenotypes as a result of mutations in non-targeted genes. Effective transfection and Cas9 cleavage will greatly increase chances of generating knockouts. Alternatively, transfecting the cells directly with commercially sourced sgRNA/Cas9 ribonucleoprotein complexes32,33 may expedite the protocol and lower off-target effects.
Dot blots are well-suited to assess protein knockouts caused by the small genomic alterations due to NHEJ. Since only a single sgRNA/Cas9 construct is necessary, this approach may be the quickest in obtaining null lines, and the blot procedure circumvents additional steps, expediting the screening. The normalized immunoblot protein signal serves as a reliable predictor of successful validation (Figure 2B). This protocol is ideal for generation of knockouts with minimal genomic perturbation. Alternatively, this method of screening can be adapted to probe for protein addition or modification, such as transgenic proteins or protein tags. However, this screening method requires an antibody with high specificity to the protein of interest, as cross-reactivity leads to high background in dot blots. This method is naturally limited to mutations targeting protein coding regions, as the final output depends on the presence/absence of a protein product. It is important to also note that mutations caused by NHEJ can lead to a variety of protein outcomes, including a protein with constitutively active, partial or dominant negative function, or a functional protein lacking the detection antibody epitope. To minimize these issues, it is helpful to target the Cas9 cut as early into the coding region as possible, in order to increase the chances of generating a fully non-functional protein. Additionally, targeting genomic regions that are less accessible to the Cas9 machinery can produce low deletion yields in the absence of selection. Finally, knockouts that confer a selective disadvantage may be difficult to obtain by this method for similar reasons.
Using homology-directed repair mechanisms together with CRISPR/Cas9 cleavage allows for much larger and more specific genomic manipulations, which can include both large scale deletions (delineated by two sgRNA cleavages) and insertions. Since two cleavage events and a precise repair process need to take place, integration of a drug resistance marker substantially increases knockout yields using this method. Although dot blot detection can also be used, screening by colony PCR is more generally applicable to accurately detect correct integration/knockout candidates with such large genomic perturbations. Further, testing by PCR allows for identification of clones with imperfect and incomplete integrations, which can provide insight into the extent and nature of the HDR process itself. Additionally, the introduced marker can be subsequently removed (by transient Cre expression or addition in membrane-permeable form), leaving a minimal, 34 bp deletion scar (loxP site) and permitting future re-use of the selection marker. The flexibility of this approach increases with the use of multiple resistance markers, allowing for the generation of multiple cumulative protein knockouts. It is important when applying this method that the absence of product is further validated, for example by RT-qPCR of the expected transcript. Since this screen can be antibody independent, this method is easily applied to protein knockouts without a robust antibody, as well as to detect changes that do not fall in protein coding genes.
The authors have nothing to disclose.
The authors would like to acknowledge Gissell Sanchez, Megan Lee, and Jason Estep for experimental assistance, and Weifeng Gu and Xuemei Chen for sharing reagents.
Competent E. coli cells | |||
plasmid prep kit | |||
pSpCas9(BB) plasmid | Addgene | 42230 | Ran et al 2013, cloning sgRNAs |
BbsI enzyme | ThermoFisher | FD1014 | Ran et al 2013, cloning sgRNAs |
T7 DNA ligase | NEB | M0318L | Ran et al 2013, cloning sgRNAs |
Tango Buffer | ThermoFisher | BY5 | Ran et al 2013, cloning sgRNAs |
PlasmidSafe exonuclease | Epicentre | E3105K | Ran et al 2013, cloning sgRNAs |
Q5 hot start high fidelity polymerase | NEB | M0494A | HA amplification |
pUC19-BsaI | Modified pUC19 plasmid, mutated existing BsaI site and inserted two outward facing BsaI sites after BamHI/EcoRI digestion | ||
pGolden-Neo | Addgene | 51422 | Resistance cassette |
pGolden-Hygro | Addgene | 51423 | Resistance cassette |
BsaI enzyme | NEB | R3535S | Homology arm contruction |
T7 DNA ligase | NEB | M0318L | |
PlasmidSafe exonuclease | Epicentre | E3105K | |
Toothpicks | bacterial PCR | ||
Taq polymerase | bacterial colony PCR | ||
HEK293 human cells | |||
DMEM | Corning | 10-013-CV | |
FBS | Corning | MT35010CV | |
Penicillin-Strep (opt.) | Gibco | 15140-122 | |
6 well plates | BioLite | 12556004 | |
TransIT-LTI Transfection Reagent | Mirus | MIR2300 | for lipofection only |
Opti-MEM | ThermoFisher | 31985062 | for lipofection only |
G418 (Neomycin) | Sigma Aldrich | A1720-5G | |
Hygromycin | Sigma Aldrich | H3274-250MG | |
96 well plates | ThermoFisher | 12556008 | |
Passive Lysis Buffer, 5x | Promega | ||
1xSDS loading buffer | recipe decribed in protocol | ||
Nitrocellulose Membrane | Bio-Rad | 162-0115 | |
TBST | recipe decribed in protocol | ||
Dehydrated milk | |||
SuperSignal West Dura Extended Duration Substrate | ThermoFisher | 34075 | for HRP-conjugated secondary antibodies |
Extracta DNA prep for PCR | Quantabio | 95091-025 | |
KOD polymerase | Novagen | 71316 |