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Research Techniques Made Simple: Delivery of the CRISPR/Cas9 Components into Epidermal Cells

      CRISPR/Cas9 technology is a powerful tool used to alter the genetic landscape of various hosts. This has been exemplified by its success in the transgenic animal world where it has been utilized to develop novel mouse lines modeling numerous disease states. The technology has helped to develop both in vitro and in vivo systems that simulate diseases within the fields of epithelial biology, skin cancer biology, dermatology, and beyond. Importantly, the delivery of the single-guide RNA/Cas9 editing complex to the host cell is key for its success. In this paper, we discuss the various methods that have been utilized as delivery techniques for CRISPR/Cas9 components, the benefits and pitfalls of each, and how successful they have been at genetically modifying epidermal cells. In addition, we acknowledge recent advances in the field of dermatology that have harnessed these methods to better understand epidermal biology, identify potential therapeutic targets, or serve as novel methods to treat disease states.

      Abbreviations:

      AAVV (adeno-associated viral vector), HPK (human primary keratinocyte), kb (kilobase), KC (keratinocyte), LVV (lentiviral vectors), RDEB (recessive dystrophic epidermolysis bullosa), SC (stem cell), SE (skin equivalent), sgRNA (single-guide RNA), TIDE (tracking of indels by decomposition)

      Introduction

      CRISPR and Cas9 provide the science community with a faster, cheaper, and more efficient method to edit genomes than previously existing techniques. This technology was adapted from a genome-editing system in bacteria used to respond to invading viruses (
      • Doudna J.A.
      • Charpentier E.
      Genome editing. The new frontier of genome engineering with CRISPR-Cas9.
      ). In nature, the CRISPR/Cas9 system uses excised pieces of viral DNA to combat reinfection from the same virus by producing RNA segments (single-guide RNA [sgRNA]) that target the viral genome for modification. The combination of sgRNA and Cas9 nuclease recognizes cDNA sequences in a viral genome and then inactivates viral genes through DNA double-stranded breaks, thus preventing replication. In the laboratory, researchers create synthetic pieces of sgRNA that can specifically bind to a sequence of interest in an organism’s genome. sgRNA/Cas9 complexes facilitate targeted DNA double-stranded breaks, which activates the cell’s DNA repair mechanisms to introduce genetic modification. This can be accomplished by insertions, deletions, or replacements with a customized DNA sequence in the target gene. Some of the uses of this system in epidermal biology, including therapeutic development and lentiviral-based genomic screens, have been discussed in previous Research Techniques Made Simple articles (
      • Guitart Jr., J.R.
      • Johnson J.L.
      • Chien W.W.
      Research techniques made simple: the application of CRISPR-Cas9 and genome editing in investigative dermatology.
      ;
      • Otten A.B.C.
      • Sun B.K.
      Research techniques made simple: CRISPR genetic screens.
      ). Importantly, the utility of this technology in genome editing was recently recognized by the Nobel Prize in Chemistry, which was awarded to Emmanuelle Charpentier and Jennifer Doudna for their pioneer work in the CRISPR/Cas9 field.

      Summary Points

      Advantages

      • CRISPR/Cas9 is a highly efficient and precise genome-editing tool.
      • CRISPR/Cas9 does not require enzyme engineering because the Cas9 enzyme remains the same for all targeting sequences. Only the single-guide RNA (sgRNA) sequence needs to be customized.
      • A single CRISPR/Cas9 editing event can target multiple loci by the addition of several sgRNAs.
      • CRISPR/Cas9 can be used to create tissues and mouse models that mimic genetic disorders or diseases for scientists to investigate potential therapeutic treatments.
      • Gene functions and molecular pathways can be interrogated using CRISPR/Cas9 genome editing.

      Limitations

      • Undesired gene modifications can occur owing to the cell’s own repair system attempting to correct double-stranded breaks that result from Cas9 cleavage.
      • Off-target effects resulting from nongene of interest sequence modification by CRISPR/Cas9 hinders its application for clinical treatment.
      • CRISPR/Cas9 has the potential of eliciting an immune response causing difficulty with repeat treatments. In addition, it can cause targeted cells to be attacked by the immune system.
      Cas9 and sgRNA can be delivered to cells in two forms: either encoded by plasmids (DNA) or in RNP complexes, which is a combination of recombinant Cas9 protein and sgRNA. Several methods of delivering these components to epithelial cells have been successfully used for genome editing, including viral and polymer-based platforms as well as physical methods (i.e., electroporation or microinjection). Plasmid-based delivery of sgRNA and Cas9 can be utilized by all delivery techniques, whereas RNP complexes are only able to be delivered by polymer-based and physical methods because these are combined in vitro before cell delivery. The four most employed methods for the delivery of sgRNA and Cas9 into epidermal cells are discussed in the following sections (Figure 1).
      Figure thumbnail gr1
      Figure 1Different delivery systems utilized for CRISPR/Cas9 gene editing. This image depicts how each of the CRISPR/Cas9-based delivery methods are developed. (a) Multiple plasmids are necessary to create viral vectors encoding both sgRNA and Cas9, which are then purified from transfected cells. (b) Either plasmids or RNP—a protein/RNA complex of Cas9 and sgRNA—are formulated into cationic vectors (the example here is a cationic lipid). No previous steps (outside of generating RNP complexes) are necessary for electroporation and/or microinjection of target cells. (c, d) The route (infection or internalization) that is used by the different delivery methods and what form of sgRNA and Cas9 the cell is exposed to. (e) All of the methods have the same outcome: generation of a gene-edited (red gene) cell of interest lacking the targeted protein (in green). AAVV, adeno-associated viral vector; LVV, lentiviral vector; sgRNA, single-guide RNA.

      Adeno-associated viral vector‒mediated delivery

      Adeno-associated viruses can be engineered to generate recombinant particles that are unable to cause active infection yet deliver genes of interest to target cells. There are three plasmids needed to generate a functional adeno-associated viral vector (AAVV): Rep-Cap plasmid, helper plasmid, and transfer plasmid. The Rep-Cap plasmid contains essential genes for the AAVV life cycle and capsid proteins (necessary for particle formation), and the helper plasmid encodes genes necessary for AAVV replication. These plasmids can be purchased from Addgene (Watertown, MA). Finally, the transfer plasmid provides the gene of interest, which would be Cas9 nuclease and/or sgRNA (
      • Naso M.F.
      • Tomkowicz B.
      • Perry 3rd, W.L.
      • Strohl W.R.
      Adeno-associated virus (AAV) as a vector for gene therapy.
      ). This plasmid needs to either be synthesized containing both sgRNA- and Cas9-encoding sequences (GeneWiz, South Plainfield, NJ; Thermo Fisher Scientific, Waltham, MA; etc) or constructed using molecular biology techniques (PCR, restriction digestion, ligation, bacterial expansion). The three plasmids are transfected into the human embryonic kidney 293 cell line to produce AAVV particles containing sgRNA and Cas9 (
      • Naso M.F.
      • Tomkowicz B.
      • Perry 3rd, W.L.
      • Strohl W.R.
      Adeno-associated virus (AAV) as a vector for gene therapy.
      ). An alternative option is to have AAVVs commercially made containing sgRNA and Cas9 components, which can be accomplished by multiple companies (Creative Biolabs, Shirley, NY; VectorBuilder, Chicago, IL; and others). These particles are then used to infect target cells, which on expressing the virally encoding sgRNA and Cas9 components are targeted for gene modification.
      There are several advantages of using AAVV to deliver sgRNA and Cas9 components. First, AAVVs have shown an appreciable safety profile in a wide range of animal models and human clinical trials (
      • Xu C.L.
      • Ruan M.Z.C.
      • Mahajan V.B.
      • Tsang S.H.
      Viral delivery systems for CRISPR.
      ). For example, AAVV does not typically integrate into the host cell genome, but when it does, this only occurs in mtDNA or in a specific location on chromosome 19. These locations are considered safe and unable to mediate tumorigenesis. AAVV is less immunogenic than other viruses, which allows it to be used for repeat treatments because it avoids neutralization by the immune system. In addition, AAVV can deliver stable sgRNA and Cas9 expression because the virus remains episomal, thereby persisting long-term in nondividing cells (
      • Xu C.L.
      • Ruan M.Z.C.
      • Mahajan V.B.
      • Tsang S.H.
      Viral delivery systems for CRISPR.
      ). In actively dividing cells, such as epithelial cells, this advantage is lost because AAVV DNA is not replicated along with host cell DNA. The disadvantage of AAVV is that its genetic packaging size is limited to approximately 4.7 kilobase (kb), which restricts single AAVV capsids from containing large genes. To address this limitation, dual-AAVV systems have been created in which one vector delivers Cas9 and another delivers the sgRNA; however, this system reduces the probability of delivering both Cas9 and sgRNA into the same cell, which can result in poor genome editing efficiencies. Because the commonly used Cas9 from Streptococcus pyogenes is large (∼4.2 kb), small orthologs of Staphylococcus aureus Cas9 (∼3.2 kb) and Str. thermophilus Cas9 (∼3.3 kb) have been designed so that they can be packaged into a single AAVV with sgRNA (
      • Lau C.H.
      • Suh Y.
      In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease.
      ) (Table 1).
      Table 1The Beneficial and Detrimental Characteristics of the Different Delivery Systems for CRISPR/Cas9 and Examples of Reported Editing Efficiencies in Epidermal Cells
      FeaturesAAVV or AdenovirusLVV-Mediated DeliveryCationic VectorsPhysical Method (Electroporation and/or Microinjection)
      Pros
      • Good safety profile
      • Low immunogenicity
      • Stable transgene expression (due to episomal persistence)
      • Infects a broad range of cells
      • Already used in patient clinical trials for gene editing
      • Large packaging size
      • Able to edit multiple genes and provide gene insertions with one vector
      • High infection efficiency
      • Stable transgene expression (integrated)
      • Already used in patient clinical trials for gene editing
      • Preferred method for large scale CRISPR screens
      • Large packaging size
      • Low immunogenicity
      • Neutralizes the anionic charge of sgRNA and Cas9
      • Easy and cheap production
      • Used mainly for in vitro studies for gene editing
      • High target specificity
      • No size delivery restrictions
      • Host-range independent
      • Used mainly in vitro and in animal studies for gene editing
      Cons
      • Small packaging size
      • Unable to edit multiple sites with one vector
      • Complicated production
      • Robust immunogenicity
      • Integrates randomly into the host genome, increasing the chance of off-target effects
      • Complicated production
      • Constant expression of sgRNA and Cas9 can increase off-target effects
      • Potential cellular toxicity
      • Low delivery efficiency
      • Potential cell damage or death
      • Nonspecific transport of molecules into and out of the cell
      • Not applicable to some cells and tissues with high resistance to electric pulsation

        For microinjection only:

      • Limitations on the number of cells that can be injected
      • Time consuming
      Cells targeted for genetic modification (observed editing efficiency or description)Human iPSC lines from epidermal progenitors (16.7–56.7%) (
      • Sebastiano V.
      • Zhen H.H.
      • Haddad B.
      • Bashkirova E.
      • Melo S.P.
      • Wang P.
      • et al.
      Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa.
      )

      HPKs (25–76%) (
      • Benati D.
      • Miselli F.
      • Cocchiarella F.
      • Patrizi C.
      • Carretero M.
      • Baldassarri S.
      • et al.
      CRISPR/Cas9-mediated in situ correction of LAMB 3 gene in keratinocytes derived from a junctional epidermolysis bullosa patient.
      )
      HPKs (35–58%) (
      • Fenini G.
      • Grossi S.
      • Contassot E.
      • Biedermann T.
      • Reichmann E.
      • French L.E.
      • et al.
      Genome editing of human primary keratinocytes by CRISPR/Cas9 reveals an essential role of the NLRP1 inflammasome in UVB sensing.
      )

        Lipofectamine 2000

      • HPKs (protein level reduction) (
        • Liu Y.C.
        • Cai Z.M.
        • Zhang X.J.
        Reprogrammed CRISPR-Cas9 targeting the conserved regions of HPV6/11 E7 genes inhibits proliferation and induces apoptosis in E7-transformed keratinocytes.
        )
      Abbreviations: AAVV, adeno-associated viral vector; HPK, human primary keratinocyte; iPSC, induced pluripotent stem cell; KC, keratinocyte; LVV, lentiviral vector; RDEB, recessive dystrophic epidermolysis bullosa; sgRNA, single-guide RNA.
      The primary author of the referenced studies is provided.
      An AAVV-based delivery of sgRNA and Cas9 showed success as a potential therapeutic intervention for patients suffering from the genetic skin disease, recessive dystrophic epidermolysis bullosa (RDEB). To correct RDEB, AAVV containing sgRNA and Cas9 were used to edit induced pluripotent stem cell (SC) lines (derived from epidermal cells) from patients with RDEB lacking functional COL7A1 in an attempt to restore the expression of the protein. After successful correction of COL7A1, cells were differentiated into keratinocytes (KCs) and used to form skin equivalents (SEs). These genetically corrected SEs showed normal growth patterns during stratification and restoration of COL7A1 expression at the basement membrane (
      • Sebastiano V.
      • Zhen H.H.
      • Haddad B.
      • Bashkirova E.
      • Melo S.P.
      • Wang P.
      • et al.
      Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa.
      ).

      Lentiviral vector‒mediated delivery

      Lentiviral vectors (LVVs) are the primary method for delivering integrated and stably expressed Cas9 and sgRNA to target cells for genetic manipulation. To generate LVV, four plasmids (or three plasmids if using second-generation constructs) are transfected into the 293T cell line to assemble viral particles. These plasmids include a pseudotyping plasmid (which determines host cell tropism), a transfer plasmid (includes both sgRNA and Cas9), and packaging plasmids (includes structural proteins and reverse transcriptase and/or integrase) (
      • Ryø L.B.
      • Thomsen E.A.
      • Mikkelsen J.G.
      Production and validation of lentiviral vectors for CRISPR/Cas9 delivery.
      ). Similar to AAVV, these plasmids can be purchased from Addgene and have to either be synthesized or constructed by molecular biology techniques (transfer plasmid). Lentiviral particles are purified from transfected cells and then used to transduce cells of interest. Alternatively, LVVs can be directly made from similar companies that produce AAVVs (Creative Biolabs, VectorBuilder, and others). When lentiviral particles infect the target cell, viral RNA, including sgRNA and Cas9, is reverse transcribed and then integrated into the genome by viral integrase. When these two components are expressed successfully, the gene of interest is targeted for modification.
      LVV-mediated delivery is advantageous owing to the large packaging size (9.7 kb) of the viral genome. The additional packaging space can contain multiple sgRNAs for genome editing or insertion sequences into targeted genetic loci, which increase the number of genes able to be modified with one infection and editing capabilities, respectively. Because LVVs facilitate the integration of sgRNA and Cas9 into infected cells, this ensures the most long-term expression of the various delivery vectors. However, LVV delivery suffers from multiple drawbacks. LVVs are robustly immunogenic, which negates multiple uses in the same host (
      • Follenzi A.
      • Santambrogio L.
      • Annoni A.
      Immune responses to lentiviral vectors.
      ). Finally, LVVs can randomly integrate into the host genome, increasing the chance of off-target effects, including gene inactivation and tumorigenesis (
      • Xu C.L.
      • Ruan M.Z.C.
      • Mahajan V.B.
      • Tsang S.H.
      Viral delivery systems for CRISPR.
      ) (Table 1).
      A recent example of LVV-mediated sgRNA and Cas9 delivery showed its use in understanding the signaling pathways necessary for epidermal responsiveness to inflammatory environments. Specifically, how deletion of NLRP1 altered the response of human primary KCs (HPK) to UVR was tested. The loss of this protein resulted in reduced responsiveness of HPKs to UV exposure as measured by inflammasome formation and cytokine secretion (
      • Fenini G.
      • Grossi S.
      • Contassot E.
      • Biedermann T.
      • Reichmann E.
      • French L.E.
      • et al.
      Genome editing of human primary keratinocytes by CRISPR/Cas9 reveals an essential role of the NLRP1 inflammasome in UVB sensing.
      ).

      Cationic vectors

      There are two categories of cationic vectors that can be used for sgRNA and Cas9 delivery: cationic polymer based and cationic lipid based. Plasmids encoding Cas9 and sgRNAs or sgRNA/Cas9 RNPs can be formulated into particles made out of cationic polymers, such as polyethyleneimine, or out of cationic lipids, such as Lipofectamine, for delivery into target cells. The positively charged cationic particles interact with the target cell membrane delivering the sgRNA and Cas9–encoding plasmids or RNP complexes into cells. Cells that have successfully taken up plasmids then produce Cas9 protein and sgRNA, which form complexes and edit the genetic material of the cell.
      The advantages of nonviral vectors include their cationic nature, which facilitates enhanced uptake and delivery of anionic sgRNA and Cas9 to the negatively charged cell. In addition, these vectors have no size limit for sgRNA and Cas9 delivery, have a reduced or nonhazardous nature, and are both simple and cheap to make. However, the cationic nature of these vectors can elicit toxicity in target cells, and some cells are inefficient at internalizing cationic particles (
      • Li L.
      • Hu S.
      • Chen X.
      Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities.
      ) (Table 1).
      Cationic vectors were recently used to develop an in vitro model for the skin disease, harlequin ichthyosis. This disease is known to arise by a mutation in the ABCA12 gene, so to model this in vitro, a combination of the cationic vectors, FuGENE 6 and HyperFect, were used to deliver sgRNA and Cas9 to an immortalized KC line (N/TERT-1) to inactivate ABCA12 (
      • Enjalbert F.
      • Dewan P.
      • Caley M.P.
      • Jones E.M.
      • Morse M.A.
      • Kelsell D.P.
      • et al.
      3D model of harlequin ichthyosis reveals inflammatory therapeutic targets.
      ;
      • Smits J.P.H.
      • Niehues H.
      • Rikken G.
      • van Vlijmen-Willems I.M.J.J.
      • van de Zande G.W.H.J.F.
      • Zeeuwen P.L.J.M.
      • et al.
      Immortalized N/TERT keratinocytes as an alternative cell source in 3D human epidermal models.
      ). Successful editing of ABCA12 was measured by a lack of protein expression. This edited cell line was then confirmed to demonstrate many of the phenotypes of harlequin ichthyosis, including dysregulated KC differentiation and lipid reorganization as well as elevated cytokine production (
      • Enjalbert F.
      • Dewan P.
      • Caley M.P.
      • Jones E.M.
      • Morse M.A.
      • Kelsell D.P.
      • et al.
      3D model of harlequin ichthyosis reveals inflammatory therapeutic targets.
      ).

      Electroporation and microinjection

      Physical methods for the delivery of sgRNA and Cas9 include the following techniques: microinjection and electroporation. Microinjection is based on direct injection of sgRNA and Cas9 components into embryos or cultured cells, whereas electroporation uses an electric field to generate pores in the cell membrane, enabling plasmids or sgRNA/Cas9 RNP complexes to enter the nucleus (
      • Li L.
      • Hu S.
      • Chen X.
      Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities.
      ).
      These methods are widely used because of their high reproducibility, simplicity, and absence of the limitations associated with viral and/or cationic vectors. Specifically, there is no limitation on the amount and size of DNA delivered to a cell. Because these techniques circumvent cellular membranes, they are not typically limited to certain cell types. The drawbacks associated with these techniques include the potential for irreversible cell damage and nonspecific transport of molecules into and out of the cell. Electroporation can result in cell death owing to sustained permeabilization of the membrane and, as a consequence, loss of cellular integrity. Some cells and tissues are also not suitable for this technique owing to their high resistance to electric pulsation (
      • Fajrial A.K.
      • He Q.Q.
      • Wirusanti N.I.
      • Slansky J.E.
      • Ding X.
      A review of emerging physical transfection methods for CRISPR/Cas9-mediated gene editing.
      ). Microinjection is a time-consuming procedure, which limits the number of cells that can be treated during a single editing event (
      • Fajrial A.K.
      • He Q.Q.
      • Wirusanti N.I.
      • Slansky J.E.
      • Ding X.
      A review of emerging physical transfection methods for CRISPR/Cas9-mediated gene editing.
      ) (Table 1).
      Electroporation has been used to develop novel therapies by inserting genes into epidermal cells with sgRNA and Cas9. A unique treatment for diabetes and obesity was developed by modifying murine KCs to express glucagon-like peptide 1. This was done by electroporating primary KCs with plasmids encoding sgRNA, Cas9, and the glucagon-like peptide 1 expression cassette. Successfully edited KCs were observed to secrete the protein into cell supernatants. Genetically modified cells were then engrafted on mice, which were subsequently fed a high-fat diet. Animals receiving modified cells demonstrated resistance to weight gain and reduced insulin resistance compared with controls (
      • Yue J.
      • Gou X.
      • Li Y.
      • Wicksteed B.
      • Wu X.
      Engineered epidermal progenitor cells can correct diet-induced obesity and diabetes.
      ).

      CRISPR/Cas9-editing efficiencies of epidermal cells from different delivery methods

      The plasmids encoding sgRNA and Cas9 or RNP complexes (recombinant Cas9 protein loaded with sgRNA) can be used to edit genomic DNA through the delivery processes described earlier (Figure 1). Importantly, both of these forms of sgRNA and Cas9 have limitations. Formulating plasmids into viral vectors is both time consuming and requires extensive molecular biology practices, but is able to deliver sustained gene-editing capabilities because Cas9 and sgRNA are continually produced by the infected cell. Delivering plasmids directly to cells through either polymer-based platform or physical manipulations results in transient expression of sgRNA and Cas9 because the cell will lose the plasmid as it divides. With regards to therapeutic treatment of patients suffering from genetic disorders, viral platforms are likely to be the chosen delivery method owing to sustained expression and targeted (viral specificity for certain cells) delivery compared with those of nonviral methods. Alternatively, RNPs are simple to assemble, especially because many companies now provide recombinant Cas9 protein and synthetic sgRNA (Synthego, Redwood City, CA; Thermo Fisher; Canopy Biosciences, St. Loius, MO; etc). RNPs can be formulated into polymers or directly delivered to cells through the physical methods discussed earlier. This form of sgRNA and Cas9 delivery is the most transient because there is no continual production of the components as occurs from infection or plasmid delivery.
      Studies have utilized the different techniques mentioned earlier to deliver sgRNA and Cas9 to epidermal cells with varying degrees of success. Multiple groups have used viral vector‒based delivery platforms for the gene editing of primary KCs. For example, AAVVs were used to deliver sgRNA and Cas9 to induced pluripotent SC lines derived from HPK. By selecting clones from the initial editing reaction, they demonstrated a 16.7% and 56.7% editing efficiency for the targeted gene using Sanger sequencing from two different cell lines (
      • Sebastiano V.
      • Zhen H.H.
      • Haddad B.
      • Bashkirova E.
      • Melo S.P.
      • Wang P.
      • et al.
      Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa.
      ). In addition, HPKs have been successfully transduced with a sgRNA and Cas9–containing adenovirus. Using four different doses, the authors observed editing efficiencies ranging from 25% to 76% using insertions or deletions, that is, indel) formation in the targeted sequence by analysis of genomic DNA from transduced cells (
      • Benati D.
      • Miselli F.
      • Cocchiarella F.
      • Patrizi C.
      • Carretero M.
      • Baldassarri S.
      • et al.
      CRISPR/Cas9-mediated in situ correction of LAMB 3 gene in keratinocytes derived from a junctional epidermolysis bullosa patient.
      ). Alternatively, sgRNA and Cas9 has been delivered to HPKs by LVVs. Editing efficiency was quantified by protein expression (western blot) and a genomic cleavage assay of the edited population of cells. This delivery technique demonstrated editing efficiencies ranging from 35% to 58% from the genomic cleavage assay (
      • Fenini G.
      • Grossi S.
      • Contassot E.
      • Biedermann T.
      • Reichmann E.
      • French L.E.
      • et al.
      Genome editing of human primary keratinocytes by CRISPR/Cas9 reveals an essential role of the NLRP1 inflammasome in UVB sensing.
      ) (Table 1).
      Studies utilizing cationic vectors for sgRNA and Cas9 delivery have demonstrated similar levels of success in editing primary KCs. One group compared the cationic lipid, Lipofectamine CRISPRMAX (Thermo Fisher Scientific), with electroporation for delivering sgRNA/Cas9 protein complexes into HPK. Using the same genomic cleavage assay mentioned earlier, Lipofectamine- and electroporation-based delivery methods resulted in 14% and 32% of indel formation in the targeted gene, respectively (
      • Yu X.
      • Liang X.
      • Xie H.
      • Kumar S.
      • Ravinder N.
      • Potter J.
      • et al.
      Improved delivery of Cas9 protein/gRNA complexes using Lipofectamine CRISPRMAX.
      ). Another example of cationic vector delivery used the cationic lipid, Lipofectamine 2000, to transfect HPKs with a plasmid containing both sgRNA and Cas9. Gene inactivation in targeted cells was then observed by changes in protein expression. No genetic analysis of editing efficiency was presented in this study (
      • Liu Y.C.
      • Cai Z.M.
      • Zhang X.J.
      Reprogrammed CRISPR-Cas9 targeting the conserved regions of HPV6/11 E7 genes inhibits proliferation and induces apoptosis in E7-transformed keratinocytes.
      ). In addition, a combination of the cationic vectors, FuGENE 6 and HyperFect transfection reagents, have been used to target genes for modification in N/TERT-1 cells. Again, gene inactivation was detected by western blot analysis of the targeted protein with no genome editing efficiency assessed (
      • Enjalbert F.
      • Dewan P.
      • Caley M.P.
      • Jones E.M.
      • Morse M.A.
      • Kelsell D.P.
      • et al.
      3D model of harlequin ichthyosis reveals inflammatory therapeutic targets.
      ).
      The main physical method used to deliver sgRNA/Cas9 protein complexes to epidermal cells is electroporation. One group tested gene-editing efficiency in electroporated HPKs by gel electrophoresis comparing nonedited with edited gene bands. This method demonstrated efficiencies ranging from 55% to 81% with different sgRNA pairs (
      • Gálvez V.
      • Chacón-Solano E.
      • Bonafont J.
      • Mencía Á.
      • Di W.L.
      • Murillas R.
      • et al.
      Efficient CRISPR-Cas9-mediated gene ablation in human keratinocytes to recapitulate genodermatoses: modeling of netherton syndrome.
      ). Another study delivered sgRNA/Cas9 complexes through electroporation to HPKs from a patient with RDEB to restore the expression of COL7A1. Using the tracking of indels by decomposition (TIDE) analysis, the authors observed editing efficiencies ranging from 69% to 77% (
      • Kocher T.
      • March O.P.
      • Bischof J.
      • Liemberger B.
      • Hainzl S.
      • Klausegger A.
      • et al.
      Predictable CRISPR/Cas9-mediated COL7A1 reframing for dystrophic epidermolysis bullosa.
      ). TIDE analysis is a web-based algorithm that compares individual Sanger sequencing reads from wild-type and DNA-edited cells, enabling the calculation of gene editing efficiency on the basis of the number of mutant to wild-type sequences from targeted cells (
      • Brinkman E.K.
      • Chen T.
      • Amendola M.
      • van Steensel B.
      Easy quantitative assessment of genome editing by sequence trace decomposition.
      ). Electroporation has also been used to deliver a plasmid containing sgRNA and Cas9 to primary mouse KCs. Unfortunately, no genetic editing efficiency was determined because the authors used a selectable marker to enrich for cells successfully targeted by their CRISPR/Cas9 system (
      • Yue J.
      • Gou X.
      • Li Y.
      • Wicksteed B.
      • Wu X.
      Engineered epidermal progenitor cells can correct diet-induced obesity and diabetes.
      ) (Table 1).
      These studies suggest that all the methods (viral, nonviral, and physical) of sgRNA and Cas9 delivery are capable of editing genes in KCs to varying degrees. Unfortunately, the method in which editing efficiency is reported across studies has not been standardized, and therefore, comparisons between different methods are difficult to make. Using analyses, such as TIDE, to assess the editing efficiency (by insertions and/or deletions) of CRISPR/Cas9 in skin cells, comparisons between each delivery method can be assessed. Importantly, many methods exist to address off-target events by CRISPR/Cas9 editing, including computational models (predictive) and whole-genome sequencing (
      • Zhang X.H.
      • Tee L.Y.
      • Wang X.G.
      • Huang Q.S.
      • Yang S.H.
      Off-target effects in CRISPR/Cas9-mediated genome engineering.
      ). None of these methods were reported in the studies discussed earlier. By assessing off-target mutations in future studies, the skin biology community would have a more comprehensive understanding of what delivery method minimizes this occurrence in skin cells. The lack of editing efficiency measurements and off-target events limits the possibility of identifying the most robust method to genetically modify epidermal cells, but does suggest that the toolbox of techniques available to epithelial biologists for CRISPR/Cas9-based genetic manipulation is extensive.

      Limitations and future directions

      This article explains different sgRNA and Cas9 delivery systems and examples of how they have been utilized in recent epidermal research. In addition, reported editing efficiencies of epidermal cells for each technique are listed. The findings from contemporary research highlights that CRISPR/Cas9 is a promising technique to validate the function of genes and their mechanistic roles in skin-specific pathways or skin-related illnesses. However, this technology still has imperfections as a genome-editing tool. Some limitations include off-target gene editing, undesired deletions or additions, possible tumorigenesis, and immunogenicity in patients receiving CRISPR/Cas9 treatments. In addition, optimization of editing efficiency in future studies will diminish the need for selective markers and/or clonal selection techniques, thus improving the speed of generation and quality of edited cell populations for both research and intervention purposes. New studies are currently focused on developing ways to overcome each of these limitations to increase the efficiency and specificity of CRISPR/Cas9-based genome modification, thereby opening new avenues for scientists to understand the function of genes in skin diseases and investigate potential therapeutic interventions for dermatological conditions.

      Conflict of Interest

      The authors state no conflict of interest.

      Multiple Choice Questions

      • 1.
        Which method of single-guide RNA (sgRNA) and Cas9 delivery most likely results in significant off-target effects?
        • A.
          Microinjection
        • B.
          Adeno-associated viral vectors (AAVVs)
        • C.
          Lentiviral vectors (LVV)
      • 2.
        Which of these methods of sgRNA and Cas9 delivery traditionally results in high cell death?
        • A.
          Cationic polymers
        • B.
          Electroporation
        • C.
          AAVVs
      • 3.
        Which method requires multiple vectors to deliver both the sgRNA and Cas9 protein owing to small packaging size restrictions?
        • A.
          AAVVs
        • B.
          Cationic polymers
        • C.
          LVV
      • 4.
        What is the function of Cas9 nuclease?
        • A.
          It contains a complementary sequence to bind the gene of interest.
        • B.
          It cuts DNA, causing a double-stranded break in a chromosome.
        • C.
          It generates sgRNA.
      • 5.
        Which method is most suitable for overcoming charge-based issues with delivery of sgRNA and Cas9 components?
        • A.
          Electroporation
        • B.
          LVV
        • C.
          Cationic vectors

      Acknowledgments

      We acknowledge the University of Rochester and the National Institutes of Allergy and Infectious Disease (U01AI152011) for salary support (MGB) and the LEO Foundation (LF18068) for support to JPHS. We would like to acknowledge the use of BioRender for figure development. The companies referred to in the manuscript are purely based on our positive experiences with their products and are by no means an endorsement.

      Author Contributions

      Conceptualization: HS, MGB; Supervision: MGB; Visualization: HS, MGB; Writing - Original Draft Preparation: HS, MGB, JPHS, EHVDB; Writing - Review and Editing: MGB, JPHS, EHVDB

      Supplementary Material

      Detailed Answers

      • 1.
        Which method of single-guide RNA (sgRNA) and Cas9 delivery most likely results in significant off-target effects?
      • CORRECT ANSWER: C. lentiviral vectors (LVV).
      • Microinjection does not require integration of sgRNA and Cas9 components, and adeno-associated viral vector (AAVV) rarely integrates, but when it does, only specific locations are utilized, which are safe and unable to promote tumorigenesis. LVVs randomly integrate into the host’s genome and, as a result, can have substantial off-target effects if they disrupt essential genes (cell death) during integration or in the worst-case scenario can promote cancer formation.
      • 2.
        Which of these methods of sgRNA and Cas9 delivery traditionally results in high cell death?
      • CORRECT ANSWER: B. Electroporation.
      • Electroporation facilitates the delivery of sgRNA/Cas9 complexes by enhancing membrane permeability through pore formation. Cells that are unable to close the pores that are formed during the process ultimately die, and in some cell types, this can be a substantial percentage of the culture.
      • 3.
        Which method requires multiple vectors to deliver both the sgRNA and Cas9 protein owing to small packaging size restrictions?
      • CORRECT ANSWER: A. AAVVs.
      • Owing to the small size of AAVV, it is only able to incorporate genes up to 4.7 kb (Cas9 is 4.1 kb) in size, which limits the ability to package both the Cas9 protein and sgRNA target sequences. As a result, multiple AAVVs containing either the Cas9 protein or the sgRNA need to be delivered to the same cell, which can impede genome editing efficiency.
      • 4.
        What is the function of Cas9 nuclease?
      • CORRECT ANSWER: B. It cuts DNA, causing a double-stranded break in a chromosome.
      • The Cas9 nuclease is guided to the target sequence by a sgRNA, and then, it cuts the gene of interest to facilitate genetic modification by the cell’s own repair mechanisms. sgRNA is a piece of RNA that can be prepared synthetically or by host nucleases (not Cas9).
      • 5.
        Which method is most suitable for overcoming charge-based issues with delivery of sgRNA/Cas9 components?
      • CORRECT ANSWER: C. Cationic vectors.
      • Cationic vectors are able to neutralize the anionic nature of the sgRNA/Cas9 complex owing to the positive charge of the polymer and/or lipid. This allows for more efficient delivery to the negatively charged cell.

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