This protocol describes the procedure for genome editing in mouse bone marrow-derived macrophages using Cas9-sgRNA ribonucleoprotein complexes assembled in vitro and delivered by electroporation.
Bone marrow-derived macrophages (BMDMs) from mice are a key tool for studying the complex biology of tissue macrophages. As primary cells, they model the physiology of macrophages in vivo more closely than immortalized macrophage cell lines and can be derived from mice already carrying defined genetic changes. However, disrupting gene function in BMDMs remains technically challenging. Here, we provide a protocol for efficient CRISPR/Cas9 genome editing in BMDMs, which allows for the introduction of small insertions and deletions (indels) that result in frameshift mutations that disrupt gene function. The protocol describes how to synthesize single-guide RNAs (sgRNA-Cas9) and form purified sgRNA-Cas9 ribonucleoprotein complexes (RNPs) that can be delivered by electroporation. It also provides an efficient method for monitoring editing efficiency using routine Sanger sequencing and a freely available online analysis program. The protocol can be performed within 1 week and does not require plasmid construction; it typically results in 85% to 95% editing efficiency.
Macrophages are innate immune cells that play critical roles in tissue repair and immunity1,2. Immortalized macrophage cell lines, such as mouse RAW 264.7 cells or human THP-1 cells, have several beneficial characteristics, including robust growth and ease of gene disruption by delivering vectors for RNA interference or CRISPR/Cas93,4. However, oncogenic transformation dramatically alters their physiology, which results in the aberrant activation of some pathways and muted responses of others5,6. Primary bone marrow-derived macrophages (BMDMs) more closely recapitulate in vivo macrophage physiology, but remain challenging to genetically manipulate due to the low efficiency of both plasmid transfection and viral transduction in these primary immune cells7,8. Thus, more efficient methods for disrupting gene function are needed.
CRISPR/Cas9 genome editing is a powerful tool for genetic manipulation across a range of biological systems, including mammalian cells9,10,11,12. The Streptococcus pyogenes Cas9 protein efficiently and specifically cleaves double-stranded DNA when complexed with a sequence-specific guide RNA. DNA repair through the non-homologous end joining (NHEJ) of the cleaved DNA results in small insertions or deletions (indels) that create frameshift mutations. In early studies, Cas9 and sgRNAs were delivered through plasmid or lentiviral vectors, which are effective delivery methods for many cell lines9,10. However, primary cells and, in particular, primary immune cells are often refractory to these methods due to the low efficiency of vector delivery by transfection or transduction. Subsequently, methods have been developed to generate sgRNA-Cas9 complexes in vitro and to deliver them via electroporation, and these methods have achieved high efficiency in a variety of cell types13,14. The results have suggested the possibility of using this approach to carry out genome editing in primary macrophages.
Here, we provide a protocol for using sgRNA-Cas9 ribonucleoprotein complexes (RNPs) to carry out genome editing in primary BMDMs. It contains steps to mitigate the activation of the immune sensors present in primary immune cells and results in up to 95% editing at targeted loci with minimal toxicity. This protocol also includes workflows to evaluate editing efficiency using routine polymerase chain reaction (PCR) and Sanger sequencing, followed by in silico analysis by Tracking of Indels by Decomposition (TIDE)15, a well-validated online software tool.
1. sgRNA design
NOTE: This step describes selection of the target sequences and design of the sgRNAs. It is helpful to design guides that are in the first large coding exon, so that any translated protein is disrupted early in the open reading frame. It is also helpful to select target sequences that lie within the same exon, as this will streamline the analysis of the editing efficiency (step 6). The examples of genome editing provided with this protocol used sgRNAs targeting the first exon of the Src gene and the Cblb gene, as well as in the non-coding Rosa26 locus of the mouse genome.
2. sgRNA synthesis
NOTE: This step describes how to synthesize sgRNAs using PCR to generate a template for in vitro transcription (IVT), and then purify the sgRNA using spin columns (Figure 1A). Custom synthetic sgRNAs are commercially available through several vendors as an alternative to PCR/IVT.
3. Preparation for electroporation
NOTE: All steps should be performed in a laminar flow hood to avoid contamination. This protocol uses a commercially available electroporation system (see Table of Materials) with 10 µL tips.
4. RNP assembly
5. RNP delivery by electroporation
6. Assessing the editing efficiency
NOTE: Most editing is complete after 48 h.
The IVT template is a 127 bp PCR product (Figure 1B). The full-length IVT product is a 98 nt RNA, which migrates similarly to a 70 bp double-stranded DNA fragment (Figure 1C).
After electroporation, the cells should be >90% viable, with a total cell count of >70% of the starting cell number. The resulting pool of mutant cells should have a diverse set of indels, starting near the Cas9 cleavage site. The analysis of the targeted genes by PCR and Sanger sequencing should show multiple nucleotides at each position downstream of the Cas9 cleavage site (Figure 2).
Figure 1: Overview of the sgRNA-Cas9 editing process. (A) Schematic for the guide design, sgRNA generation, and Cas9-sgRNA delivery. (B) The PCR product from IVT for Src sgRNAs 1, 2, and 3 resolved on a 2% agarose gel. The arrow indicates the correct 127 bp PCR products. (C) The RNA products following IVT for Src sgRNAs 1, 2, and 3 resolved on a 2% agarose gel. The brackets indicate the correct sgRNA products. The variable migration of the sgRNA is due to RNA secondary structure. This figure is reprinted from the first author's master's thesis16. Please click here to view a larger version of this figure.
Figure 2: High editing efficiency achieved for multiple targeted genes. (A) Schematic of PCR and Sanger sequencing primers. (B) Schematic of the workflow for TIDE to assess the editing efficiency. (C) Representative Sanger sequencing chromatogram of the ROSA26 locus from BMDMs electroporated with a control non-targeting sgRNA-Cas9 RNP (top) and ROSA26-specific sgRNA (bottom); the Cas9 cleavage site is highlighted. The TIDE output (right) with the calculated editing efficiency and the percentage of sequences harboring the indicated number of indels. (D,E) Sanger sequencing chromatograms of the (D) Src and Cblb genes in the edited BMDMs. This figure is reprinted from the first author's master's thesis16. Please click here to view a larger version of this figure.
Figure 3: Moderate increase in editing efficiency due to the commercial electroporation enhancer. The BMDMs were electroporated with low-efficiency guides to evaluate the effect of a commercial editing enhancer. Prior to electroporation, 1 µL of the enhancer was added to the assembled RNPs to a final concentration of 4 µM. The editing efficiency of the indicated Src sgRNA was evaluated using TIDE. This figure is reprinted from the first author's master's thesis16. Please click here to view a larger version of this figure.
Tool Name | URL | ||
Synthego – CRISPR Design Tool | https://www.synthego.com/products/bioinformatics/crispr-design-tool | ||
The Broad Institute – CRISPick | https://portals.broadinstitute.org/gppx/crispick/public | ||
Tracking of Indels by Decomposition (TIDE) | http://shinyapps.datacurators.nl/tide/ |
Table 1: URLs of online tools.
Primer Name | Sequence | ||
Guide-Specific Primer | ggatcctaatacgactcactatag[N20]gttttagagctagaa | ||
Guide-Specific Primer – Cbl-b Guide 3 | ggatcctaatacgactcactatagAAAATATCAAGTATATACGGgttttagagctagaa | ||
Guide-Specific Primer – Cbl-b Guide 4 | ggatcctaatacgactcactatagGGTAAAATATCAAGTATATAgttttagagctagaa | ||
Guide-Specific Primer – Rosa | ggatcctaatacgactcactatagCTCCAGTCTTTCTAGAAGATgttttagagctagaa | ||
Guide-Specific Primer – Scramble | ggatcctaatacgactcactatagGCACTACCAGAGCTAACTCAgttttagagctagaa | ||
Guide-Specific Primer – Src Guide 2 | ggatcctaatacgactcactatagTCACTAGACGGGAATCAGAGgttttagagctagaa | ||
Guide-Specific Primer – Src Guide 5 | ggatcctaatacgactcactatagCAGCAACAAGAGCAAGCCCAgttttagagctagaa | ||
Guide-Specific Primer – Src Guide 6 | ggatcctaatacgactcactatagAGCCCAAGGACGCCAGCCAGgttttagagctagaa | ||
T7 Reverse Long Universal Primer | aaaaaagcaccgactcggtgccactttttcaagttgataacggactagccttattttaacttgctatttctagctctaaaac | ||
Universal Forward Amplification Primer | ggatcctaatacgactcactatag | ||
Universal Reverse Amplification Primer | aaaaaagcaccgactcgg |
Table 2: Oligonucleotides used in the PCR to generate the template for the IVT of the sgRNA. The 20 nucleotide target sequences for gene-specific primers are capitalized.
BMDM Growth media. Store at 4 C. | |||
DMEM | |||
Fetal bovine serum | 0.1 | ||
L-glutamine | 0.2 M | ||
MCSF supernatant from 3T3-MCSF Cells** | 0.1 | ||
Sodium pyruvate | 11 mg/mL | ||
Lysis Buffer. Store at 4 C. | |||
2-mercaptoethanol (add immediately prior to use) | 0.01 | ||
MgCl2 | 5 mM | ||
Tris | 20 mM | ||
Triton-X 100 | 0.005 | ||
**3T3-MCSF cells are grown in DMEM+10% FCS. Supernatent with MCSF is harvested on the 5th day after reaching 100% confluence. As an alternative 10ng/ml recombinant MCSF can be used in lieu of conditioned media |
Table 3: Compositions of the media and buffers.
Supplementary File 1: Raw sequencing file for ROSA_ Mock Please click here to download this File.
Supplementary File 2: Raw sequencing file for ROSA_TargettingGuide Please click here to download this File.
Supplementary File 3: Raw sequencing file for ScrambleGuide Please click here to download this File.
Supplementary File 4: Raw sequencing file for SrcG5+SrcG6 Please click here to download this File.
Supplementary File 5: Raw sequencing file for CBLB_Mock Please click here to download this File.
Supplementary File 6: Raw sequencing file for CBLB_TargetingGuide Please click here to download this File.
Supplementary File 7: Raw sequencing file for Mock_Guide2Locus Please click here to download this File.
Supplementary File 8: Raw sequencing file for Mock_Guide6Locus Please click here to download this File.
Supplementary File 9: Raw sequencing file for SrcGuide2_Enhancer Please click here to download this File.
Supplementary File 10: Raw sequencing file for SrcGuide2_NoEnhance Please click here to download this File.
Supplementary File 11: Raw sequencing file for SrcGuide6_Enhancer Please click here to download this File.
Supplementary File 12: Raw sequencing file for SrcGuide6_NoEnhancer Please click here to download this File.
Genome editing using electroporated Cas9-sgRNA complexes allows effective disruption of gene function in BMDMs. The editing efficiency varies by the target sequence and gene. Typically, four to five sgRNAs are generally screened to identify one that is highly active. Some loci have lower editing efficiencies, most likely due the chromatin structure. In these cases, several modifications can be made to increase the editing efficiency. Co-delivery of two active sgRNAs to the same exon results in improved editing for some genes. However, when two guides are co-transfected, we have observed that TIDE may lose accuracy. Therefore, alternative techniques to assess editing, such as Western blotting, may be required. In addition, the inclusion of a commercially available NHEJ enhancer often increases the editing efficiency by ~20% (Figure 3).
This approach has several advantages. The direct delivery of sgRNA-Cas9 to cells does not require the time-consuming steps of plasmid construction or lentiviral vector production to transduce BMDMs. The process of synthesizing sgRNA, electroporation, and generating mutant cells can be completed in 1 week. This technique can also be used with BMDMs derived from genetically modified mice to create double-mutant cells. Although chemical methods exist for transfecting primary immune cells with siRNA or mRNA17, these chemical methods are significantly less effective than electroporation at delivering Cas9-sgRNA complexes to immune cells18. There are several types of electroporation devices available commercially. These devices are anticipated to work similarly for the delivery of Cas9-sgRNA complexes, although the voltage parameters likely need to be optimized for each individual device. While this protocol has been used primarily on mouse BMDMs, in limited experiments, similar results have been obtained with rat BMDMs (unpublished data). It is possible that other types of primary macrophages, such as mouse peritoneal macrophages or alveolar macrophages, might also be amenable to this approach, though this has not yet been tested.
This method has some limitations. The number of cells produced by this protocol is somewhat limited; our standard conditions generate 4 x 105 cells per electroporation. However, it may be possible to scale up the yield significantly, as the efficiency is undiminished in a 10 µL reaction with up to 2.4 x 106 cells using the same amount of RNP (unpublished data). In addition, 100 µL electroporation tips are available. Another drawback of this method is the expense; the costs of both the electroporator and the electroporation consumables are substantial. These expenses can be significantly mitigated by reusing the tips when using different sgRNAs targeting the same gene and by using PBS instead of the proprietary buffers. While the compositions of the proprietary buffers are not known, presumably the electrolyte composition of PBS with 0.9 mM CaCl2 and 0.5 mM MgCl2 approximates the electrical conductance of the proprietary buffer. These changes reduce the cost of consumables by roughly 80% in this protocol.
There are several critical steps in the protocol, deviations from which could dramatically affect the efficiency of the gene editing. One of the potential pitfalls is that standard IVT kits, which are not designed for high yields, often produce insufficient IVT product. In addition, the use of standard polypropylene microfuge tubes instead of low-binding tubes can cause significant cell loss by adhesion. The incomplete dephosphorylation of the IVT product and the presence of residual DNA in the sgRNA preparation may result in the activation of macrophage immune sensors and subsequent toxicity. Higher or longer voltage pulses may also result in increased cell death.
In summary, this genome editing protocol, which uses electroporation to deliver Cas9-sgRNA RNPs, is an efficient method to disrupt genes in mouse BMDMs. This allows users to rapidly screen for phenotypes in primary cells that more closely recapitulate the complex biology of macrophages in vivo.
The authors have nothing to disclose.
This work was funded by the NIH grant 5R01AI144149. The schematic figures were created with BioRender.
3T3-MCSF Cell Line | Gift from Russell Vance | not applicable | |
Alt-R Cas9 Electroporation Enhancer | IDT | 1075915 | |
Ampure XP Reagent Beads | Beckman Coulter | A63880 | |
Calf intestinal alkaline phosphatase | NEB | M0525S | |
DNase | NEB | M0303S | |
DPBS +Ca/Mg (0.9mM CaCl2 and 0.5mM MgCl2) | Thermo Fisher | 14040-133 | |
DPBS -Ca/Mg | Thermo Fisher | 14190-144 | |
ExoI | NEB | M0293S | |
Fetal Calf Serum (FCS) | Corning | 35-015-CV | |
Herculase DNA polymerase & buffer | Agilent | 600677 | |
HiScribe T7 High Yield RNA Synthesis Kit | NEB | E2040S | |
LoBind conical tubes 15 mL | Eppendorf | 30122216 | |
LoBind Eppendorf tubes 2 mL | Eppendorf | 22431102 | |
NEBuffer r2.1 | NEB | B6002S | |
Neon Transfection System | Thermo Fisher | MPK5000, MPP100, MPS100 | |
Neon Transfection System 10 uL Tips | Thermo Fisher | MPK1025 or MPK1096 | |
PBS + 1mM EDTA | Lonza | BE02017F | |
Proteinase K | Thermo Fisher | EO0491 | |
rCutSmart Buffer for ExoI | NEB | B6004S | |
Ribolock | Thermo Fisher | EO0384 | |
RNA loading dye | NEB | B0363S | |
RNeasy Mini Kit | Qiagen | 74104 | |
S. pyogenes Cas9-NLS | University of California Macro Lab | not applicable | Available to non-UC investigators through https://qb3.berkeley.edu |
S. pyogenes Cas9-NLS, modified 3rd Generation | IDT | 1081059 | |
SAP | NEB | M0371S |