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. 2022 Nov 10;102(2):197–206. doi: 10.1177/00220345221128201

Efficient Gene Knockout in Salivary Gland Epithelial Explant Cultures

R Sekiguchi 1, MM Mehlferber 1,2, K Matsumoto 1, S Wang 1,3,✉
PMCID: PMC9893391  PMID: 36366748

Abstract

We have developed methods to achieve efficient CRISPR-Cas9–mediated gene knockout in ex vivo mouse embryonic salivary epithelial explants. Salivary epithelial explants provide a valuable model for characterizing cell signaling, differentiation, and epithelial morphogenesis, but research has been limited by a paucity of efficient gene perturbation methods. Here, we demonstrate highly efficient gene perturbation by transient transduction of guide RNA–expressing lentiviruses into Cas9-expressing salivary epithelial buds isolated from Cas9 transgenic mice. We first show that salivary epithelial explants can be cultured in low-concentration, nonsolidified Matrigel suspensions in 96-well plates, which greatly increases sample throughput compared to conventional cultures embedded in solidified Matrigel. We further show that salivary epithelial explants can grow and branch with FGF7 alone, while supplementing with insulin, transferrin, and selenium (ITS) enhances growth and branching. We then describe an efficient workflow to produce experiment-ready, high-titer lentiviruses within 1 wk after molecular cloning. To track transduced cells, we designed the lentiviral vector to coexpress a nuclear fluorescent reporter with the guide RNA. We routinely achieved 80% transduction efficiency when antibiotic selection was used. Importantly, we detected robust loss of targeted protein products when testing 9 guide RNAs for 3 different genes. Moreover, targeting the β1 integrin gene (Itgb1) inhibited branching morphogenesis, which supports the importance of cell–matrix adhesion in driving branching morphogenesis. In summary, we have established a lentivirus-based method that can efficiently perturb genes of interest in salivary epithelial explants, which will greatly facilitate studies of specific gene functions using this system.

Keywords: CRISPR, Cas9, lentivirus, HSP47, integrin, morphogenesis

Introduction

The mouse salivary gland is an excellent model for studying mammalian organ development. During mouse embryogenesis, an epithelial bud bulges from the oral epidermis to become surrounded by neural crest–derived mesenchyme to form the primordial salivary gland (Jaskoll et al. 2002). Between the epithelium and mesenchyme is a dense, thin layer of extracellular matrix, the basement membrane (Sekiguchi and Yamada 2018). The initial epithelial bud grows and repetitively splits into numerous buds arranged in a “bunch of grapes” appearance through branching morphogenesis, while epithelial cells differentiate into ductal, acinar, or myoepithelial cells, whose concerted actions enable saliva secretion in matured organs (Patel et al. 2006; Tucker 2007; Wang et al. 2017).

Salivary gland research has been advanced by a combination of in vivo and ex vivo approaches. Mouse gene knockout studies definitively established the importance of various signaling pathways, transcriptional factors, and extracellular matrix regulation in salivary gland development (Patel et al. 2006; Knosp et al. 2015; Chatzeli et al. 2017; Emmerson et al. 2017; Szymaniak et al. 2017; Athwal et al. 2019). On the other hand, the ex vivo organotypic culture of mouse embryonic salivary glands enabled characterizations and manipulations that are only possible outside embryos (Borghese 1950). For example, live imaging of ex vivo cultured salivary glands revealed extensive dynamics of epithelial cells and the basement membrane matrix, which catalyzed the discovery that a combination of strong cell–matrix and weak cell–cell adhesions in surface epithelial cells drives branching morphogenesis of salivary glands (Larsen et al. 2006; Wei et al. 2007; Harunaga et al. 2014; Wang et al. 2021). Furthermore, the mesenchyme encapsulating the salivary epithelium can be surgically removed to allow mesenchyme-free culture of the salivary epithelium in the presence of basement membrane extract and certain growth factors (Nogawa and Takahashi 1991). These salivary epithelial explants enabled analysis of how different growth factors affect their growth, morphogenesis, or differentiation (Steinberg et al. 2005; Nakao et al. 2017).

Small-molecule inhibitors, blocking antibodies, soluble dominant-negative receptors, and small interfering RNA (siRNA) have all been used to perturb gene functions in ex vivo cultures of salivary glands (Sakai et al. 2003; Steinberget al. 2005; Wei et al. 2007), although each has some limitations. Small-molecule inhibitors can be effective to perturb certain signaling pathways or enzymatic activities, but their performance varies depending on their efficacy and specificity. Blocking antibodies or soluble dominant-negative receptors can selectively inhibit ligand binding to surface receptors, but they are only available for a small number of targets. While siRNA-mediated gene knockdown can generalize to most genes, it comes with many disadvantages, including transient inhibition, unpredictable knockdown levels, and significant off-target effects.

Compared to siRNA-mediated gene knockdown, CRISPR-Cas9–mediated gene knockout offers persistent disruption and better specificity (Boettcher and McManus 2015). CRISPR-Cas9 is an RNA-guided DNA endonuclease that can make site-specific double-stranded DNA breaks in mammalian cells (Jinek et al. 2012; Cong et al. 2013). These DNA breaks trigger endogenous DNA repair machinery to catalyze nonhomologous end joining (NHEJ) or homology-directed repair (HDR), which can be leveraged for various purposes of genome editing. Notably, knockout of protein coding genes can be readily achieved by frameshift mutations introduced by error-prone NHEJ repair. CRISPR-Cas9 has not been used in ex vivo salivary gland cultures, partly due to a lack of an efficient delivery system for the single guide RNA (sgRNA) needed to target a specific gene.

Here, we demonstrate highly efficient CRISPR-Cas9–mediated gene knockout in ex vivo cultures of mouse embryonic salivary epithelial explants, which was achieved by transient transduction of sgRNA-expressing high-titer lentiviruses into Cas9-expressing salivary epithelial buds isolated from Cas9 transgenic mice (Platt et al. 2014). To facilitate application of this method, we developed new salivary epithelial isolation and culture techniques that greatly improved sample throughput and an efficient workflow to produce experiment-ready, high-titer lentiviruses within 1 wk after molecular cloning. Importantly, we showed that branching morphogenesis was inhibited when the β1 integrin gene (Itgb1) was targeted, indicating our lentiviral delivery system is efficient enough to produce loss-of-function phenotypes at the tissue level. This approach has broad applications to test the roles of genes that may contribute to salivary gland development or disease.

Materials and Methods

Methods for sgRNA design, plasmid cloning, lentivirus packaging, lentivirus transduction, salivary epithelial explant isolation and culture, quantitative polymerase chain reaction (qPCR), immunostaining, microscopy, image processing, analysis, and quantification are in the Appendix.

Mouse Strains

Mouse experiments were approved by the National Institute of Dental and Craniofacial Research (NIDCR) Animal Care and Use Committee (Animal Study Protocols 17-845 and 20-1040). Standard mouse housing and husbandry were provided by the NIDCR Veterinary Resource Core. Timed pregnant ICR (CD-1) outbred mice were obtained from Envigo to get wild-type embryos. The Cas9 transgenic mice (Platt et al. 2014) were obtained from the Jackson Laboratory (JAX, 026558). To generate embryos at specific gestational stages, transgenic mice 8 to 16 wk old were bred, where the next day after a vaginal plug was found was defined as embryonic day 1.

Experiment Design

This study complied with Animal Research: Reporting In Vivo Experiments (ARRIVE) 2.0. For each experiment, all submandibular salivary glands from all embryos without sex identification (mixed sex) from 1 timed pregnant mouse were used to isolate individual epithelial buds. The epithelial buds with similar sizes were selected for transduction with lentiviruses expressing a control sgRNA or an sgRNA targeting a gene of interest. The extent of branching morphogenesis or the targeted protein expression level was compared between explants grown from epithelial buds expressing the control sgRNA and those expressing the targeting sgRNA. No animals or embryos were excluded from experiments. Criteria used for excluding qPCR data points are detailed in the Appendix (see the qPCR section). Exact sample sizes are provided in the figure legends.

Data, Code, and Resource Availability

All data of this study are available in Figshare (https://doi.org/10.6084/m9.figshare.21298803). All plasmids are available in Addgene (https://www.addgene.org/browse/article/28229196/). Customized scripts and usage instructions are available from GitHub (https://github.com/snownontrace/Sekiguchi-et-al-2022). Selected step-by-step protocols can be found at https://snownontrace.github.io/.

Results

Salivary Epithelial Bud Isolation and Explant Culture in 96-Well Plates

We first optimized the isolation and culture techniques for salivary epithelial explants to increase sample throughput (Appendix Fig. 1A). Conventionally, salivary gland mesenchyme is removed from the epithelium by dissection after dispase treatment. The whole epithelial rudiment or individual epithelial buds are then embedded in high-concentration Matrigel (a basement membrane extract) that quickly solidified. In our new procedure, multiple salivary glands were triturated in bulk after dispase treatment to dissociate the mesenchyme into single cells, whereas the epithelial buds remained intact, presumably due to stronger cell–cell adhesion. Each epithelial bud was then cultured in nonsolidified Matrigel in the well of an ultra-low-attachment 96-well plate, which greatly facilitated sample handling and high-throughput imaging using microscopy systems with capabilities for automated scanning of multi-well plates.

Next, we tested several combinations of growth factors using the new culture format. We found that epithelial explants grew well with FGF7, NRG1, and insulin, transferrin, and selenium (ITS; Fig. 1A, B). The explants failed to grow with only NRG1 and ITS, supporting the critical role of FGF signaling in salivary epithelial development (Steinberg et al. 2005). Removing ITS from the combination significantly slowed explant growth, whereas removing NRG1 did not affect explant growth or budding morphogenesis (Fig. 1A, B). Thus, we chose to use the FGF7 and ITS combination for standard explant cultures.

Figure 1.

Figure 1.

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Salivary epithelial explant culture and antibiotic enrichment of lentivirus-transduced cells. (A) Phase contrast images of single-bud explant cultures at 2 d under indicated culture conditions. FGF7, 200 ng/mL. NRG1, 10 ng/mL. ITS, 10 mg/L insulin, 5.5 mg/L transferrin, and 6.7 µg/L selenium. Nonsolidified Matrigel (MG), 400 to 500 µg/mL. (B) Plot of bud number per explant under indicated culture conditions. Sample number: n = 13, 15, 11, 12, and 10 for conditions 1 to 5. (C) Schematics of the lentivirus transduction experiment. (D) Nuclear staining and segmentation of explant cultures treated with DMSO (vehicle control) or 20 µg/mL blasticidin. The NLS-mScarlet channel was subjected to a gamma of 0.5 to facilitate simultaneous display of both bright and dim nuclei. (E) Plot of percentage of transduced cells in DMSO- or blasticidin-treated explant cultures at 4 d. Error bars, standard deviation. Statistics, Tukey test (B) or Student’s t test (E). ***P < 0.001. n.s., not significant. Scale bars: (A) 100 µm, (D) 20 µm.

Streamlined Production of High-Titer sgRNA-Expressing Lentiviruses

To maximize efficiency, we tested the use of lentivirus to express sgRNAs in salivary epithelial explants. We used a previously published lentiviral vector system (Wang et al. 2021) to coexpress a nuclear fluorescent reporter with the sgRNA (NLS-mScarlet-I or NLS-tagBFP) to follow transduced cells and a blasticidin resistance gene (BlastR) to allow antibiotic selection (Appendix Fig. 1B). These vectors also enabled straightforward molecular cloning by 1-step ligation of the sgRNA oligo duplex (Appendix Fig. 1B), which could be easily adapted for parallel cloning of tens of sgRNA vectors. Using miniprep-grade plasmid DNAs, we generated experiment-ready, high-titer lentiviruses in 5 d (Appendix Fig. 1C). To estimate lentivirus titer, we used a quick commercial test to measure the test-band intensity (Appendix Fig. 1D) and compared that to a reference virus. The typical titer of sgRNA-expressing lentiviruses using this method was about 1.5 × 108 IFU/mL. After transient transduction of lentiviruses into epithelial buds, nuclear reporter expression could be detected by the next day and reached a maximum level within 2 d. About 50% of the cells in the explants expressed nuclear reporters without antibiotic selection, while this ratio was raised to 80% with antibiotic selection (Fig. 1C–E), indicating that antibiotic selection could efficiently enrich transduced cells.

Knockout of β1 Integrin Inhibits Branching Morphogenesis

For gene perturbation, we transduced sgRNA-expressing lentiviruses into Cas9-expressing salivary epithelial buds isolated from Cas9 transgenic E13 mouse embryos (Platt et al. 2014). We used an sgRNA targeting the Escherichia coli lacZ gene as the control and tested 3 targeting sgRNAs for Itgb1 (Appendix Table 1), which encodes β1 integrin that mediates cell–matrix adhesion (Kechagia et al. 2019). All sgRNA sequences were verified by Sanger sequencing (Appendix Fig. 2A).

To use qPCR to distinguish wild-type and mutant Itgb1 messenger RNAs (mRNAs), we designed qPCR primer pairs with 1 primer overlapping the CRISPR/Cas9 cut site using an established method (Fig. 2A; Li et al. 2019). These primers can efficiently and specifically amplify their targets (Appendix Fig. 2B). We found that all 3 Itgb1 sgRNAs could efficiently induce mutations of the targeted mRNAs, as shown by the reduced levels of wild-type mRNAs (Fig. 2B).

Figure 2.

Figure 2.

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Knockout of β1 integrin inhibits branching morphogenesis. (A) Schematics of quantitative polymerase chain reaction (qPCR) primers for testing single guide RNA (sgRNA) efficiencies. (B) Plot of normalized expression levels of wild-type Itgb1 messenger RNAs (mRNAs) by qPCR. The ratio of the testing mRNA to the reference Rps29 mRNA was normalized to the average value of the sg-Control group. Sample number (left to right): n = 13, 13, 13, 13, 20, 17. (C) Confocal fluorescence images of explant cultures transduced with lentiviruses expressing indicated sgRNAs and immunostained with anti-β1 integrin. Explants were fixed at 2 d posttransduction. Cyan boxes mark the single-channel magnified images. Asterisks mark cells that are negative for β1 integrin staining but expressing high (yellow asterisks) or low (cyan asterisks) levels of the sgRNA reporter. The NLS-mScarlet channel was subjected to a gamma of 0.5 to facilitate simultaneous display of both bright and dim nuclei. (D) Schematic of the quantification method (left) and plots (right) of the β1 integrin intensity across edges of adjacent transduced cells. Five edges per explant were quantified. Sample number (edges; left to right): n = 15, 15, 25, 25 (day 1); 25, 25, 20, 25 (day 2); 25, 25, 25, 45 (day 4); and 25, 25, 25, 25 (day 7). (E) Phase contrast images of explant cultures transduced with lentiviruses expressing indicated sgRNAs at 4 different time points (with blasticidin selection). (F) Plot of bud number per explant over time. Sample number (left to right): n = 19, 19, 19, 20 (day 1); 4, 5, 5, 5 (day 2); 10, 9, 10, 10 (day 3); 15, 15, 15, 15 (day 4); and 5, 5, 5, 5 (day 7). Note that only the central image plane of day 7 samples was used for bud counting, whereas all image planes were used for other time points. Error bars in all plots, standard deviation. Statistics, Tukey test (>2 groups) or Student’s t test (2 groups). *P < 0.05. **P < 0.01. ***P < 0.001. n.s., not significant. Comparisons were all made to the sg-Control group. Scale bars: 20 µm (C), 100 µm (E).

To determine whether and when sgRNA expression could lower protein expression, we performed immunostaining at 1, 2, 4, and 7 d after transduction. We found that expressing sg2- or sg3-Itgb1 reduced β1 integrin expression to background levels by day 2 (Fig. 2C, D), while a noticeable reduction was observed as early as 1 d posttransduction (Fig. 2D, Appendix Fig. 3A). On the other hand, expressing sg1-Itgb1 only marginally lowered β1 integrin expression on day 1 (Fig. 2D, Appendix Fig. 3A), but the effect reached a similar level as the other 2 sgRNAs by day 4 (Appendix Fig. 3A). Thus, different guide RNAs could have different kinetics when mediating protein reduction. Interestingly, the level of protein reduction was insensitive to the level of sgRNA expression, because cells with low or high sgRNA reporter expression reduced targeted proteins to similar levels (Fig. 2C, yellow and cyan asterisks).

We next analyzed explant growth and morphogenesis after gene disruption. All 3 Itgb1 sgRNAs strongly inhibited the increase in numbers of buds by day 3 (Fig. 2E, F, Appendix Fig. 3B). The effect of sg1-Itgb1 was less severe than with the other 2 sgRNAs, consistent with the slower kinetics of sg1-Itgb1 in reducing β1 integrin expression.

To evaluate whether this approach works for salivary epithelial explants starting from a later stage, we performed lentiviral transduction using epithelial bud clusters from E16 Cas9 embryos. We found that expressing any of the 3 Itgb1 sgRNAs was able to reduce protein expression (Fig. 3A) and inhibit budding morphogenesis (Fig. 3B). Thus, we conclude that lentivirus-mediated sgRNA delivery can robustly disrupt gene functions in salivary epithelial explants from different embryonic stages.

Figure 3.

Figure 3.

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Efficient knockout of β1 integrin in E16 salivary epithelial explants. (A) Confocal fluorescence images of E16 explant cultures transduced with lentiviruses expressing indicated single guide RNAs (sgRNAs) and immunostained with anti-β1 integrin. Explants were fixed at 4 d posttransduction. Cyan boxes mark the single-channel magnified images. The NLS-mScarlet channel was subjected to a gamma of 0.5 to facilitate simultaneous display of both bright and dim nuclei. (B) Schematic of the quantification method (upper) and plots (lower) of the β1 integrin intensity across edges of adjacent transduced cells. Five edges per explant were quantified. Sample number (edges; left to right): n = 50, 45, 30, 40. (C) Phase contrast images of explant cultures transduced with lentiviruses expressing indicated sgRNAs (with blasticidin selection; 4 d posttransduction). (D) Plot of bud ratio per explant. Sample number: n = 5 for each group. Note that only the central image plane was used for bud counting. Error bars in all plots, standard deviation. Statistics, Tukey test. **P < 0.01. ***P < 0.001. Comparisons were all made to the sg-Control group. Scale bars: 20 µm (A), 100 µm (C).

Efficient Perturbation of 2 Other Genes in Salivary Epithelial Explants

We next tested 3 guide RNAs each for 2 other genes, Itga9 and Serpinh1 (Appendix Table 1, Appendix Figs. 4A, 5A). Itga9 is the most highly expressed α integrin gene in the E13 salivary epithelium (Wang et al. 2021), whereas Serpinh1 encodes HSP47, a collagen-specific chaperone that is important for collagen biogenesis (Ito and Nagata 2017). The exceptionally high GC content of the Itga9 sgRNA-containing exon (Appendix Fig. 4B) prevented us from qPCR analysis of Itga9, whereas qPCR analysis of Serpinh1 revealed significant reduction of wild-type mRNAs when sgRNAs were expressed (Fig. 5A, B, Appendix Fig. 5B). For both Itga9 and Serpinh1, we showed that all sgRNAs were able to reduce target protein expression by immunostaining (Figs. 4A, B and 5C, D). Therefore, lentivirus-mediated sgRNA delivery in Cas9 transgenic epithelial explants can efficiently reduce protein expression for all 3 tested genes.

Figure 5.

Figure 5.

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Efficient gene perturbation of Serpinh1. (A) Schematics of quantitative polymerase chain reaction (qPCR) primers for testing single guide RNA (sgRNA) efficiencies. (B) Plot of normalized expression levels of wild-type Serpinh1 messenger RNAs (mRNAs) by qPCR. The ratio of the testing mRNA to the reference Rps29 mRNA was normalized to the average value of the sg-Control group. Sample number (left to right): n = 13, 12, 13, 12, 13, 13. (C) Widefield (upper) or maximum intensity projection of 10-µm z-range of confocal (lower) fluorescence images of single-bud explant cultures transduced with lentiviruses expressing indicated sgRNAs and immunostained with anti-HSP47 (the Serpinh1 gene encodes the HSP47 protein). EGFP was coexpressed with the Cas9 transgene. Explants were fixed at 2.5 d posttransduction. (D) Plot of mean HSP47 intensity per imaging field. Sample number: n = 7, 7, 4, 6 nonoverlapping imaging fields from 2 explants for the 4 groups. (E) Phase contrast images of explant cultures transduced with lentiviruses expressing indicated sgRNAs (with blasticidin selection; 4 d posttransduction). (F) Plot of bud number per explant. Sample number: n = 10 for each group. (G) Confocal fluorescence images of explant cultures transduced with lentiviruses expressing indicated sgRNAs and immunostained with anti–collagen IV. Explants were fixed at 4 d posttransduction. The NLS-mScarlet channel was subjected to a gamma of 0.5 to facilitate simultaneous display of both bright and dim nuclei. (H) Plot of the collagen IV intensity across edges between a transduced cell and the basement membrane. Sample number (left to right): n = 45, 20, 50, 30 edges from 3 explants for each group (5 edges per field of view). Error bars in all plots, standard deviation. Statistics, Tukey test (>2 groups) or Student’s t test (2 groups). *P < 0.05. **P < 0.01. ***P < 0.001. n.s., not significant. Comparisons were all made to the sg-Control group. Scale bars: 100 µm (C, E), 20 µm (G).

Figure 4.

Figure 4.

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Efficient and specific gene perturbation of Itga9. (A) Confocal fluorescence images of E13 explant cultures transduced with lentiviruses expressing indicated single guide RNAs (sgRNAs) and immunostained with anti-α9 integrin. Cyan boxes mark the single-channel magnified images shown above. Explants were fixed at 3 d posttransduction. The NLS-mScarlet channel was subjected to a gamma of 0.5 to facilitate simultaneous display of both bright and dim nuclei. (B) Schematic of the quantification method (upper) and plot (lower) of the α9 integrin intensity across edges of adjacent transduced cells. Sample number: n = 20 edges from 4 different explants for each group. (C) Phase contrast images of explant cultures transduced with lentiviruses expressing indicated sgRNAs (with blasticidin selection; 4 d posttransduction). (D) Plot of bud number per explant. Sample number (left to right): n = 15, 14, 14, 15. (E, H) Plot of normalized expression levels of Itga3 (E) or Itga6 (H) messenger RNAs (mRNAs) by qPCR. The ratio of the testing mRNA to the reference Rps29 mRNA was normalized to the average value of the sg-Control group. Sample number (left to right): n = 9, 9, 10, 8 (Itga3); 9, 9, 10, 10 (Itga6). (F, I) Confocal fluorescence images of explant cultures transduced with lentiviruses expressing indicated sgRNAs and immunostained with anti-α3 integrin (F) or anti-α6 integrin (I). Explants were fixed at 4 d posttransduction. Cyan arrows point to enriched α6 integrin expression along the bud periphery. The NLS-mScarlet channel was subjected to a gamma of 0.5 to facilitate simultaneous display of both bright and dim nuclei. (G, J) Schematics of the quantification method (upper) and plots (lower) of the α3 or α6 integrin intensity. The α3 integrin intensity was quantified across edges of adjacent transduced cells, whereas the α6 integrin intensity was quantified across edges of a transduced cell and the basement membrane. Five edges per explant were quantified. Sample number (edges; left to right): n = 25, 20, 20, 20 (α3 integrin); 20, 20, 25, 25 (α6 integrin). Error bars in all plots, standard deviation. Statistics, Tukey test. **P < 0.01. ***P < 0.001. n.s., not significant. Scale bars: 20 µm (A, F, I), 100 µm (C).

Unlike Itgb1, no gross phenotypes were observed when Itga9 or Serpinh1 was disrupted (Figs. 4C, D and 5E, F). We examined the expression of α3 and α6 integrins by qPCR and immunostaining, where we found no or little changes of either upon Itga9 disruption (Fig. 4E–J, Appendix Fig. 4C), suggesting the lack of phenotype when targeting Itga9 was due to the continued presence of other α integrins that could dimerize with β1 integrin. On the other hand, the lack of phenotype when targeting Serpinh1 was likely because sufficient collagen IV was provided by the Matrigel supplement. Consistent with this, immunostaining of collagen IV, the major type of collagen in the basement membrane, revealed similar collagen IV expression when Serpinh1 was targeted (Fig. 5G, H).

Discussion

In this study, we describe successful implementation of CRISPR-Cas9–mediated gene knockout in ex vivo salivary epithelial explant cultures. We achieved robust gene disruption by transient transduction of high-titer sgRNA-expressing lentiviruses into Cas9-expressing salivary epithelial buds isolated from Cas9 transgenic mice. A major advantage of gene ablation in ex vivo cultures is to bypass the need to generate knockout mice to analyze gene functions. To facilitate the application of this method, we established simplified procedures for isolating and culturing salivary epithelial buds that are both less labor-intensive and higher throughput than conventional procedures. We demonstrated successful protein reduction for 3 targeted genes and inhibited branching morphogenesis when targeting the β1 integrin gene. This approach will be broadly useful to analyze the functions of other specific genes using ex vivo cultures of various tissues.

The Efficiency of CRISPR-Cas9–Mediated Gene Disruption

While siRNA targets the mRNA, CRISPR-Cas9 targets the genomic DNA. The superior efficacy of CRIPR-Cas9–mediated gene disruption is partly because there are usually only 2 copies of genes, whereas the mRNA molecules of the same gene can number tens to thousands. Moreover, DNA disruption is heritable and thus only needs to occur once, whereas siRNAs need to constantly fight against newly transcribed mRNAs, and they become diluted as cells divide.

The efficiency of CRISPR-Cas9 can be optimized by sgRNA design. Mechanistically, gene disruption by CRISPR-Cas9 mainly comes from frameshift mutations introduced during the error-prone NHEJ repair of DNA breaks. Importantly, the error patterns of NHEJ are strongly influenced by the sequence context. Several studies have developed algorithms that predict how likely an sgRNA will cause out-of-frame mutations (Bae et al. 2014; Doench et al. 2016; Chen et al. 2019). These predictive models have been incorporated as a few efficiency and outcome scores into the intuitive web-based sgRNA design tool CRISPOR to guide the choice of sgRNAs (Concordet and Haeussler 2018).

The lentivirus-based sgRNA delivery ensures persistent sgRNA expression, which further increases the probability of gene disruption. In rare cases, the double-stranded DNA breaks might be repaired perfectly without generating mutations, but these would be cut again until some mutations are generated to prevent further cuts by the CRISPR-Cas9 enzyme. It is worth noting that the U6 promoter we use to drive sgRNA expression imposes 2 additional considerations on the sgRNA design. First, if the sgRNA does not begin with the nucleotide G, an extra G should be added to the beginning due to transcriptional start site preference of the U6 promoter (Goomer and Kunkel 1992; Ma et al. 2014). Second, certain sequence motifs at the 3′ end of sgRNA can severely lower gene knockout efficiencies (Graf et al. 2019). The sgRNAs containing these motifs are highlighted as “inefficient” when using CRISPOR for sgRNA design, and these sgRNAs should be avoided for gene disruption.

The Specificity of CRISPR-Cas9–Mediated Gene Disruption

Both siRNA and CRISPR-Cas9 have off-target effects, but off-targets of CRISPR-Cas9 can be predicted much more reliably and thus be more effectively mitigated with strategic sgRNA design (Hsu et al. 2013; Bae et al. 2014; Cradick et al. 2014; Doench et al. 2016; Chen et al. 2019). Cutting by CRISPR-Cas9 requires the protospacer adjacent motif (PAM) and is most sensitive to mismatches within 11 bp of the PAM (Jinek et al. 2012; Cong et al. 2013), but it can tolerate certain insertion/deletion mutations in the sgRNA that causes DNA or RNA bulges (Lin et al. 2014). The PAM for the Streptococcus pyogenes Cas9 (SpCas9) we use is NGG, which is relatively abundant in the coding sequence of genes. As a result, there are often tens to hundreds of sgRNAs with high-specificity scores from which to choose for any target gene. When selecting sgRNAs for gene knockout studies, it is good practice to prioritize high specificity and to use several sgRNAs for the same target gene.

Integrin-Mediated Cell–Matrix Adhesion in Branching Morphogenesis

The importance of β1 integrin in salivary epithelial branching morphogenesis has been established via antibody blocking experiments (Wei et al. 2007). β1 integrin has also been shown to mediate the assembly of human salivary stem/progenitor microstructures by siRNA-mediated knockdown (Wu et al. 2019). Our results in this study provide genetic perturbation evidence that strengthens the finding that β1 integrin is required for branching morphogenesis.

β1 integrin can dimerize with multiple types of α integrins to mediate cell–matrix adhesion. We find that disrupting α9 integrin alone does not affect branching morphogenesis, suggesting that either other types of α integrins are more important, or multiple α integrins can act redundantly to provide the necessary cell–matrix interactions for branching morphogenesis.

Applications of CRISPR-Cas9 Gene Knockout in Ex Vivo Cultures

Lentivirus-based sgRNA delivery for CRISPR-Cas9 gene knockout in ex vivo cultures can be easily adapted for analyzing gene functions at the tissue or cell level by adjusting lentivirus titers. For tissue-level phenotypes, such as explant growth and branching morphogenesis, it will be better to use the highest possible titer to transduce most cells in the explant. The penetrance of perturbation phenotypes may be further enhanced by antibiotic selection of transduced cells. For cell-level phenotypes, however, it will be better to lower the virus titer to generate mosaic knockout, so that only a small fraction of cells is perturbed to maintain the native tissue environment. For example, changes of intracellular organization or transcriptional activities during cell differentiation can be examined by immunostaining or single-molecule RNA fluorescence in situ hybridization (smFISH) in mosaic knockout explants (Raj et al. 2008; Wang 2019).

Our work focuses on epithelial cells, but lentivirus can also transduce many other cell types. One possible future application is to test whether it can mediate efficient gene knockout in mesenchymal cells, which will enable studies of specific gene functions in epithelial–mesenchymal interactions.

Author Contributions

R. Sekiguchi, contributed to conception, data acquisition and analysis, drafted and critically revised the manuscript; M.M. Mehlferber, K. Matsumoto, contributed to data acquisition, critically revised the manuscript; and S. Wang, contributed to conception and design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript. All authors gave final approval and agreed to be accountable for all aspects of the work.

Supplemental Material

sj-pdf-1-jdr-10.1177_00220345221128201 – Supplemental material for Efficient Gene Knockout in Salivary Gland Epithelial Explant Cultures
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Supplemental material, sj-pdf-1-jdr-10.1177_00220345221128201 for Efficient Gene Knockout in Salivary Gland Epithelial Explant Cultures by R. Sekiguchi, M.M. Mehlferber, K. Matsumoto and S. Wang in Journal of Dental Research

Footnotes

A supplemental appendix to this article is available online.

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: We thank members of the Cell Biology Section for helpful discussions, Kenneth Yamada and Di Wu (NIDDK) for critical reading of the manuscript, the National Institute of Dental and Craniofacial Research (NIDCR) Imaging Core for microscopy support, the NIDCR Combined Technical Research Core for Sanger sequencing, and the NIDCR Veterinary Resource Core for animal support. S. Wang is supported in part by an NIDCR K99 Pathway to Independence Award (K99 DE27982). This work is supported by the NIH Intramural Research Program (NIDCR, ZIA DE000525).

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Supplementary Materials

sj-pdf-1-jdr-10.1177_00220345221128201 – Supplemental material for Efficient Gene Knockout in Salivary Gland Epithelial Explant Cultures
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Supplemental material, sj-pdf-1-jdr-10.1177_00220345221128201 for Efficient Gene Knockout in Salivary Gland Epithelial Explant Cultures by R. Sekiguchi, M.M. Mehlferber, K. Matsumoto and S. Wang in Journal of Dental Research

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