CRISPR/Cas9 Ribonucleoprotein Nucleofection for Genome Editing in Primary Human Keratinocytes: Knockouts, Deletions, and Homology‐Directed Repair Mutagenesis

Antibiotic resistance-based selection methods for screening recipient cells result in low efficiency in HKs due to their short lifespan.

“Additionally, the antibiotic resistance–based selection methods associated with the screening of recipient cells results in low efficiency considering the short lifespan of HKs (Fenini et al., 2018).”

• cites Fenini et al., 2018 sourceDisputed

  In “Establishment of a protocol for stable genetic modification of keratinocytes” (page unavailable), the paper says antibiotic selection in co-culture enabled efficient modification of human primary keratinocytes, contrary to the claimed low efficiency. It notes that these cells stop proliferating after a few passages under certain culture conditions and that prolonged puromycin selection harms survival, but does not attribute low selection efficiency to their short lifespan. (LLM, conf=0.86)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

The use of an RNP complex is associated with reduced experimental time because the pre-assembled complex starts cutting immediately without waiting for plasmid transcription and translation.

“Moreover, the use of an RNP complex is associated with reduced experimental time, as the pre‐assembled complex is introduced into the cell nucleus and starts cutting immediately, without having to wait for the plasmid to be transcribed and the Cas9 protein to be translated (Kim et al., 2014).”

• cites Kim et al., 2014 sourceSupported

Constitutive expression of Cas9 and gRNA from lentiviral vectors increases the risk of off-target effects.

“Moreover, the constitutive expression of both Cas9 protein and gRNA increases the risk of off‐target effects (Lino et al., 2018; Papapetrou & Schambach, 2016).”

• cites Lino et al., 2018 sourcePartly supported

  The “Lentivirus (LV) and adenovirus (AdV)” section (page unavailable) says viral-payload integration can increase expression and off-target effects. The “Cas9 activity” section (page unavailable) also links excessive sgRNA to off-target effects. Neither passage specifically says that constitutive expression of both Cas9 and gRNA is what increases the risk. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites Papapetrou & Schambach, 2016 sourceCould not verify

  The genomic safe harbors section discusses risks from lentiviral vector integration, including disruption of nearby genes, but not off-target effects caused by constitutive Cas9 and gRNA expression. The section on targeting predetermined safe harbors names CRISPR-Cas9 as a targeting tool without addressing that risk. Searches for constitutive Cas9/gRNA expression and prolonged expression causing off-target cleavage found no supporting passage in our copy. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

This protocol has been tested on HKs cultured in EpiLife Medium with no differences in genome editing efficiency compared to culture on feeder cells.

“However, this protocol has also been tested on HKs cultured in EpiLife Medium (Gibco, MEPI500CA) with no differences in genome editing efficiency.”

The best efficiency/survival ratio for primary human keratinocytes was achieved using the DS138 nucleofection program.

“We found that the best efficiency/survival ratio for primary HKs was achieved using the DS138 program.”

Nucleofection of Cas9/sgRNA ribonucleoprotein complexes efficiently generates deletion of specific genomic regions in primary human keratinocytes.

“By nucleofecting ribonucleoprotein complexes, we efficiently generate KO cells as well as deletion of specific genomic regions.”

• Also stated in: § DELETION OF SPECIFIC GENOMIC REGION USING RNPs VIA NUCLEOFECTION · plain

  “This method allows up to 90% of targeted genomic deletion in primary HKs in only 3 days without clonal selection.”

• Also stated in: § DELETION OF SPECIFIC GENOMIC REGION USING RNPs VIA NUCLEOFECTION · boosted

  “Representative DNA agarose gel showing efficient deletion of a DNA segment in primary HKs transfected with two Cas9/sgRNA RNP complexes (EDITED) compared to Cas9 protein only (CTR).”

Nucleofection and plating of 1 × 10^5 cells in one well of a 24-well plate or 2 × 10^5 cells in a 12-well plate did not cause loss of cell viability.

“We have nucleofected and plated 1 × 105cells in one well of a 24‐well plate or plated 2 × 105 cells in a 12‐well plate (Corning, 3513) without loss of cell viability.”

Using sgRNA1+sgRNA2+Cas9 results in less efficient deletion of the region of interest than using two independent RNP complexes.

“In fact, in the first case, despite the reduction in cost by using a smaller amount of Cas9 protein, we found less efficient deletion of the region of interest, probably due to competition between sgRNAs for the Cas9 protein.”

Nucleofection of Cas9/sgRNA ribonucleoprotein complexes efficiently generates gene knock-out cells in primary human keratinocytes.

“By nucleofecting ribonucleoprotein complexes, we efficiently generate KO cells as well as deletion of specific genomic regions.”

• Also stated in: § KNOCK‐OUT GENERATION BY INDEL MUTATION IN PRIMARY HUMAN KERATINOCYTES USING NUCLEOFECTION OF RIBONUCLEOPROTEIN (RNP) COMPLEX · plain

  “To achieve high CRISPR/Cas9 genome editing efficiency (>90%) in a small number of HKs without clonal selection of the targeted cells, this protocol employs transient nucleofection of an RNP complex consisting of Cas9 protein and sgRNA containing chemical modifications to stabilize the RNA and increase its resistance to nuclease activity.”

Using an immortalized HK cell line (N/TERT2G), a pre-screening approach was set up to sub-select potential mutant clones by designing an HDR donor oligo carrying a restriction site sequence that results in a silent mutation.

“Using an immortalized HK cell line (N/TERT2G), we set up a pre‐screening approach to preliminary sub‐select potential mutant clones by designing an HDR donor oligo carrying a restriction site sequence that results in a silent mutation.”

For the squamous cell carcinoma cell line SCC13, the ED138 nucleofection program could improve nucleofection efficiency without excessively affecting cell viability.

“However, we noticed that for other epithelial cell lines, in particular the squamous cell carcinoma cell line SCC13 (Rheinwald & Beckett, 1981), the program ED138 could be used to improve nucleofection efficiency without excessively affecting cell viability.”

Using two independent RNP complexes formed by sgRNA1+Cas9 and sgRNA2+Cas9 provides the highest editing efficiency for deletion of a specific genomic region.

“We found that using two independent RNP complexes formed by sgRNA1+Cas9 and sgRNA2+Cas9 provides the highest editing efficiency.”

Doubling the amount of Cas9 protein (sgRNA1+sgRNA2+2XCas9) still results in inefficient deletion of the region of interest.

“In the second case, despite doubling the amount of Cas9 protein that could theoretically bind the sgRNAs with the same efficiency, we still found an inefficient deletion of the region of interest.”

PCR for the genomic region of interest, followed by enzymatic digestion and agarose gel electrophoresis, allows simple and clear pre-selection of putative genome-edited clones.

“PCR for the genomic region of interest, followed by enzymatic digestion and agarose gel electrophoresis, allows simple and clear pre‐selection of putative genome‐edited clones.”

Control cells nucleofected with Cas9 protein alone produce a clean PCR band corresponding to the amplicon containing the sequence of interest.

“Control cells that were uniquely nucleofected with Cas9 protein produce a clean band corresponding to the PCR amplicon containing the sequence of interest.”

The protocol achieves 50% to 60% efficiency of the desired modification.

“With this protocol, we have achieved 50% to 60% efficiency of the desired modification.”

No significant differences in editing efficiency or survival are observed between positive and negative strands of Alt-R HDR Donor Oligos.

“We observed no significant differences in efficiency or survival between positive and negative strands of Alt‐R™ HDR Donor Oligos.”

Control cells nucleofected with Cas9 protein alone retain high expression of the protein of interest.

“As expected, the control cells still have a high expression level of the protein of interest, as Cas9 could not proceed with the cut without the gRNA.”

CRISPR/Cas9 bulk targeted cells show very low levels of the protein of interest.

“On the other hand, CRISPR/Cas9 bulk targeted cells show very low levels of the protein of interest.”

Comparable genome editing efficiency is achieved using both 1 × 10^5 and 2 × 10^5 cells without scaling up Cas9 and sgRNA volumes.

“We were able to achieve comparable genome editing efficiency using both 1 × 105 and 2 × 105 cells without scaling up Cas9 and sgRNA volumes.”

The protocol achieves nucleotide substitution with impressive efficiency compared to previous technologies.

“Although the mechanism of HDR is statistically less efficient than NHEJ, this protocol outlines an optimized method to achieve nucleotide substitution with impressive efficiency as compared to previous technologies.”

• AssumptionRests on an assumption: HDR is statistically less efficient than NHEJ.

This pre-screening method reduces the number of clones undergoing DNA sequencing.

“This method reduces the number of clones undergoing DNA sequencing.”

Competition between sgRNAs for the Cas9 protein probably explains the reduced deletion efficiency when sgRNA1+sgRNA2+Cas9 is used.

“In fact, in the first case, despite the reduction in cost by using a smaller amount of Cas9 protein, we found less efficient deletion of the region of interest, probably due to competition between sgRNAs for the Cas9 protein.”

The described protocol can be applied for KO generation in N/TERT2G or in cancer cell lines of epithelial origin (such as SCC13, SCC12, or A431).

“The described protocol can be also applied for KO generation in N/TERT2G (Dickson et al., 2000) or in cancer cell lines of epithelial origin (such as SCC13, SCC12, or A431).”

Nucleofection is the most efficient non-viral transfection approach for primary cells.

“nucleofection has been reported as the most efficient non‐viral transfection approach for primary cells (Aluigi et al., 2006).”

• cites Aluigi et al., 2006Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

The presence of a band corresponding to the control band in RNP-treated cells is probably due to the absence of a selection method to isolate edited cells.

“Again, the presence of a band corresponding to the control band is probably due to the fact that no selection method was used to isolate the edited cells.”

A single-clone selection step is essential to identify clones carrying the mutation of interest.

“However, a single‐clone selection step is essential to identify the clones carrying the mutation of interest.”

The protocols described can be adopted in different laboratories to manipulate human keratinocytes with high efficiency.

“The protocols described here can be adopted in different laboratories to manipulate HKs with high efficiency.”

The described methods require small volumes of reagents.

“The main advantages of the methods described here are the high efficiency, the reduced number of cells used for each reaction, and the small volume of reagents needed, which improve the cost‐efficiency ratio.”

The described methods improve the cost-efficiency ratio.

“The main advantages of the methods described here are the high efficiency, the reduced number of cells used for each reaction, and the small volume of reagents needed, which improve the cost‐efficiency ratio.”

The described methods have high efficiency as a main advantage.

“The main advantages of the methods described here are the high efficiency, the reduced number of cells used for each reaction, and the small volume of reagents needed, which improve the cost‐efficiency ratio.”

The described methods use a reduced number of cells per reaction.

“The main advantages of the methods described here are the high efficiency, the reduced number of cells used for each reaction, and the small volume of reagents needed, which improve the cost‐efficiency ratio.”

The higher band intensity of the shorter amplicon with the deletion compared to the control band indicates highly efficient genomic deletion.

“The higher band intensity of the shorter amplicon with the deletion compared to the control band indicates a highly efficient genomic deletion.”

Single-clone isolation is required for site-specific mutagenesis experiments.

“For this reason, single‐clone isolation is required for site‐specific mutagenesis experiments.”

The faint band corresponding to the desired molecular weight in bulk targeted cells is due to the absence of a selection method to isolate edited cells.

“The presence of a faint band corresponding to the desired molecular weight is due to the fact that no selection method was used to isolate the edited cells.”