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CRISPR Cas9 Gene Knockout/Knock-in Case Studies

45 min Read
Overview

Thanks to CRISPR's unparalleled versatility it is now more affordable than ever before to perform precise genetic edits to almost any part of the genome! This addition of the CRISPR Cas9 knowledge base offers two case studies in which the CRISPR Cas9 system was used to knockout or knock-in a gene in a specific cell line. The two case studies presented were performed using abm's CRISPR Stable Knockout Cell Line Generation Service and Custom CRISPR HDR Template Service.

CRISPR Stable Knockout Cell Line Generation

Using CRISPR to develop a biallelic LIF Knockout in Mouse Colon Carcinoma Cells



Summary

  • LIF locus in a Mouse Colon Carcinoma Cell Line was knocked out using CRISPR targeted genome editing.
  • Surveyor assay and sequencing results showed genome editing.
  • After monoclonal selection biallelic knockout was confirmed by sequencing.

Phase 1: Cas9 and sgRNA Delivery

  • Three sgRNA were designed against mouse LIF locus (Mus musculus, NM_008501). Software analysis was performed to ensure the sgRNA had no predicted off target binding sites. The selected sgRNA design was then cloned into the pLenti-U6-sgRNA-SFFV-Cas9-2A-Puro All-in-One lentivector (Figure 1).
  • Recombinant Lentiviruses were packaged using abm’s second generation Lentiviral packaging system. A multiplicity of infection (MOI) of 5 was used to transduce the cells.
CRISPR Cas9 - sgRNA Lentivector


Figure 1 – pLenti-U6-sgRNA-SFFV-Cas9-2A-Puro lentivector is an all-in-one vector for co-expressing sgRNA and Cas9 in mammalian cells. Expression of sgRNA is driven by the U6 promoter, a strong constitutive Pol III promoter. An SFFV promoter drives expression of the Cas9-2A-Puro cassette. By using the Cas9-2A-Puro cassette, cells can be directly screened for expression of Cas9, as they will be resistant to Puromycin.


Phase 2: First Round of Colony Screening for Edited Clones

  • Cell colonies are isolated after puromycin selection. Genomic DNA was extracted and the surveyor assay was performed to confirm genomic editing of the LIF locus.
  • A single band in a surveyor assay at the wild-type (WT) size indicates no editing has occurred; two smaller bands (that sum to the length of the WT) indicate editing has taken place.
  • The surveyor assay (Figure 2) indicated that Colony 3 and 6 were edited; colony 2 was not edited; and colony 1 was inconclusive.
CRISPR Cas9 - Surveyor Assay Results


Figure 2 – The surveyor assay indicated that Colony 3 and 6 were edited; colony 2 was not edited; and colony 1 was inconclusive.


Phase 3: Sequence Analysis of the Edited Colonies

  • PCR products from Colonies 3 and 6 were further analyzed via Sanger Sequencing to determine the nature of the knockout (Figure 3).
  • For colony 3 only one mutant sequence was detected, indicating that these cells are likely only heterozyotic knockouts. In colony 6 two different mutant sequences were detected.
CRISPR Cas9 - Sanger Sequencing Analysis 1

Figure 3 – For colony 3 only one mutant sequence was detected, indicating that these cells are likely only heterozyotic knockouts. In colony 6 two different mutant sequences were detected.


Phase 4: Second Round of Selection for Monoclonal Biallelic Knockout Clones

  • Colony 6 was serial diluted into 96 well plates for monoclonal selection. Genomic DNA was extracted from these clones (i.e. 6a, 6b..), PCR amplified, cloned and sequenced.
  • Of the colony 6 clones, sequencing showed that only clone 6a had a frameshift mutation in both alleles (Figure 4). A frameshift mutation disrupts the open reading frame, resulting in nonsense mediated decay of mRNA transcript.
CRISPR Cas9 - Sanger Sequencing Analysis 2

Figure 4 – Clones 6a, 6b and 6d all showed biallelic editing. Only clone 6a had frame shift mutations in both alleles. No WT sequences were detected in all subclones.


CRISPR Cas9 - Sanger Sequencing Analysis 2 3

Figure 5 – Further sequencing of 6a confirmed biallelic knock-out. No WT sequences were detected.


Phase 5: Confirmation of Knockout by Next Generation Amplicon Sequencing

  • With next generation sequencing hundreds of thousands of alleles can be sequenced at once, resulting in a more robust dataset. By contrast Sanger sequencing is only feasible for 1-100 clones and therefore it can miss a large proportion of the population.
  • Next generation sequencing was performed at each stage of selection to evaluate knockout (Figure 6). Before editing, only WT sequences were observed. After the first round of selection colony 6 showed a mixture of edited (70%) and WT (30%) sequences. Finally after monoclonal selection, clone 6a showed only edited sequences with no WT alleles present.

CRISPR Cas9 - Next Generation Sequencing Validation

Figure 6 – Next Generation Sequencing for CRISPR Knockout screening. A) Before knock-out only WT sequences are detected. B) After Cas9 and sgRNA delivery, the first round of selection shows a mixed distribution of indel and WT sequences. C) After the second round of selection only knockouts remain.

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CRISPR Stable Knockin Cell Line Generation

Using CRISPR to Knock-in Red Fluorescent Protein (RFP) gene into Human Embryonic Kidney Cells at the AAVS1 Safe Harbor Site



Summary

  • An expression cassette containing RFP and puromycin resistance genes (pAAVS1-RFP-DNR) was knocked into the AAVS1 Safe Harbor site in HEK293 cells using CRISPR targeted genome editing via the HDR pathway. Gene insertion at a Safe Harbour site allows stable gene expression without any adverse effects on the fitness of the engineered cells.
  • Genomic PCR confirmed Knocked–in RFP integration at AAVS1 Safe Harbor locus.
  • RFP expression was confirmed in cells by fluorescence microscopy.
CRISPR Cas9 - HDR Pathway


Figure 7 – CRISPR Knock-in requires expression of Cas9 and sgRNA to produce a double-stranded break. The repair template, shown here as pAAVS1-RFP-DNR, is used by the cell to repair the break using homologous recombination. The desired gene and selection marker (RFP and puromycin) included between the homology arms on the repair template will be integrated into the genome.


Phase 1: Construction and Delivery of sgRNA, Cas9 and Repair Template

  • An sgRNA was designed against the human AAVS1 Safe-harbor locus
  • Software analysis was performed to ensure the sgRNA had no predicted off target binding sites. The selected sgRNA design, along with the CMV promoter-driven Cas9 gene, was cloned into pCas-Guide to make pCas-Guide-AAVS1 (Figure 8).
  • The pAAVS1-RFP-DNR donor plasmid was designed to contain the RFP-puromycin expression cassette, flanked on either side by homology arms of 600 bp (Figure 8).
  • HEK293 cells were co-transfected with both plasmids using DNAfectin transfection reagent.
CRISPR Cas9 - AAVS1 Repair Template Vector


Figure 8 – Vector maps of pCas-Guide-AAVS1 and pAAVS1-RFP-DNR. pCas-Guide-AAVS1 is an all-in-one vector for co-expression of sgRNA and Cas9 in mammalian cells. Expression of sgRNA is driven by the U6 promoter, a strong constitutive Pol III promoter; while a CMV promoter drives the expression of the Cas9 enzyme. pAAVS1-RFP-DNR expresses puromycin resistance marker under the PGK promoter and RFP gene under the CMV promoter. The 5’ and 3’ AAVS1 homology arms (‘AAVS-Right’ and ‘AAVS-Left’) provide the cells with a template for Homology Directed Repair.


Phase 2: Dilution of the Donor Plasmid and Resistance Marker Selection

  • Transfected HEK293 cells were passaged ten times to dilute out the episomal donor vector.
  • After these passages puromycin was added to the media to select for cells with successful knock-in of the RFP-puromycin resistance cassette.
  • After 3-4 weeks of selection, >95% of HEK293 cells were expressing RFP.
CRISPR Cas9 - HEK293 Edited Cells


Figure 9 – After transfection, HEK293 cells were passaged ten times to dilute out the episomal vector, then grown in the presence of puromycin for 4 weeks. A) Cells transfected with both pCas-Guide-AAVS1 and pAAVS1-RFP-DNR were healthy after 4 weeks. B) Over 95% of these cells imaged in Figure 9 (A) expressed RFP. C) Control cells not transfected with the vectors died after puromycin treatment.


Phase 3: Confirmation of Knock-in by Genomic PCR

  • To confirm knock-in of RFP in the genomic DNA, a primer pair was designed with Primer 1 targeting the 5’ homology arm upstream of RFP and Primer 2 targeting within the RFP-Puromycin resistance cassette.
  • PCR product of 1.1 kb indicates successful knock-in at AAVS1 site; absence of PCR amplification indicates unsuccessful cassette insertion (Figure 10).
  • No PCR amplification was seen in the control cells (‘WT cell’) since Primer 2 could not anneal to the genomic DNA.
CRISPR Cas9 - Knock-in PCR Confirmation


Figure 10 – Genomic PCR was used to confirm the knock-in of RFP. In edited cells, both primer 1 and primer 2 can bind, resulting in a 1.1 kb PCR product. No PCR product is formed in WT cells as primer 2 cannot anneal to the genomic DNA.

CRISPR Bacterial Gene Knockout Case Study

CRISPR-assisted knockout of chloramphenicol resistance cassette (CAT) in E. coli.



Summary

  • A genomically encoded chloramphenicol resistance cassette (referred to as CAT - chloramphenicol acetyl transferase) was knocked out using CRISPR-assisted genome editing.
  • E. coli transformants were screened for sensitivity to chloramphenicol and correct chromosomal insertion of repair template.
  • CAT knockout was confirmed by sequencing.
We have this E. coli CRISPR Knockout experiment available as a teaching kit! Contact us for more information on how you can do CRISPR in the classroom.

Phase 1: Cas9 and sgRNA Design and Cloning

  • To improve recombination rates in bacteria, phage-derived (λ red) recombinases were employed alongside Cas9 in pCas to carry out enhanced homologus recombination (Figure 11).
  • sgRNAs were designed against the CAT gene which was previously introduced into the E. coli genome at the yeeR locus (accession number: NP_416505). The resulting sgRNAs were then cloned into pTarget (Figure 11).
  • Repair templates were designed as single-stranded oligonucleotides containing homology to the CAT gene. The repair template also contains three stop codons for the early termination of CAT and a unique restriction site for screening purposes (Figure 13). Importantly, the repair template eliminates the PAM site, preventing Cas9 re-targeting and cleavage of edited cells.
Vector maps of pCas and pTarget


Figure 11 – Vector maps of pCas and pTarget. pCas9 constitutively expresses Cas9, whereas the λ red genes are inducible. pTarget constitutively expresses the sgRNA to guide Cas9 to the target locus.


Phase 2: Preparation of λ Red-induced Electrocompetent Cells and Transformation

pCas, carrying the λ red genes and Cas9, was transformed into E. coli cells. These cells were then made electrocompetent and the λ red genes were induced prior to co-transformation of pTarget and the repair template.



Phase 3: Screening and Sequencing - Knockout of Genomically-encoded Chloramphenicol Resistance Cassette (CAT)


a. Screening for sensitivity to chloramphenicol


  • Transformants were replica picked onto kanamycin and chloramphenicol agar plates to assess sensitivity to chloramphenicol.
  • Successful knockout and inhibition of the CAT gene is indicated by growth on kanamycin plates, but no growth on chloramphenicol plates (Figure 12).
Replica plates of potential CAT knockouts


Figure 12 – Replica plates of potential CAT knockouts. Replica plates demonstrate 11/45 transformants were successfully edited (circled in red). The kanamycin plate is shown on the left, and the chloramphenicol plate on the right. Wild type controls (carrying the kanamycin plasmid and CAT gene integrated into the chromosome) are shown at the bottom of each plate.


b. Screening for correct chromosomal insertion of repair template by restriction digest


  • Successfully edited transformants can be verified by restriction enzyme digest using the unique SpeI site (Figure 13).
Schematic of CAT gene knockout using a repair template containing stop codons and SpeI site


Figure 13 – Schematic of CAT gene knock out using a repair template containing stop codons and SpeI site.


  • The target locus was PCR amplified from the chloramphenicol-sensitive colonies and then digested using SpeI and NcoI to reveal a unique digest profile (Figure 14).
Agarose gel depciting restriction digest profiles of the chloramphenicol-sensitive colonies


Figure 14 – Agarose gel depicting restriction digest profiles of the chloramphenicol- sensitive colonies. PCR products subjected to SpeI/NcoI restriction digest produces three bands for a positive clone and two bands for a negative clone. Lane 1: 100 bp Opti-DNA Marker. Lane 2-9: Colonies #1-8. Lane 10: Negative control.



c. Sequencing of Chloramphenicol-sensitive and Restriction Digest Positive Colonies

  • PCR products were subjected to Sanger sequencing to confirm correct insertion and knock out of the CAT gene (Figure 15).
Sequence alignment of CAT gene knockout colonies compared to wild type and repair template.


Figure 15 – Sequence alignment of CAT gene knockout colonies compared to wild type and repair template. The knockout insertion sequence (green) depicts the three stop codons (red) and the SpeI restriction site (underlined).

CRISPR Bacterial Gene Knock-in Case Study

CRISPR-assisted knock-in of the mCherry cassette in E. coli


Summary

  • The mCherry cassette was knocked into the chromosome using CRISPR-assisted genome editing.
  • Transformants were screened using colony PCR.
  • mCherry knock-in was confirmed by sequencing.

Phase 1: Cas9 and sgRNA Design and Cloning

  • To improve recombination rates in bacteria, phage-derived (λ red) recombinases were employed alongside Cas9 in pCas to carry out enhanced homologus recombination (Figure 16).
  • sgRNAs were designed against the yeeR locus (accession number: NP_416505). Each sgRNA was individually cloned into pTarget (Figure 16).
  • Repair templates were designed as double-stranded DNA containing the mCherry cassette flanked by homologies to the yeeR locus (Figure 17).
Cector maps of pCas and pTarget. pCas9 constitutively expresses Cas9, whereas the λ red genes are inducible.


Figure 16 – Vector maps of pCas and pTarget. pCas9 constitutively expresses Cas9, whereas the λ red genes are inducible. pTarget constitutively expresses the sgRNA to guide Cas9 to the target locus.


Phase 2: Preparation of λ Red-induced Electrocompetent Cells and Transformation

pCas, carrying the λ red genes and Cas9, was transformed into E. coli cells. These cells were then made electrocompetent and the λ red genes were induced prior to co-transformation of pTarget and the repair template.



Phase 3: Screening and Sequencing - Knock-in of a Chromosomal mCherry Cassette


a. Colony PCR Screening for Insertion of Chromosomal mCherry Cassette


  • Colonies were subjected to PCR using one primer specific to the upstream region of the integration site on the chromosome and one primer specific to the mCherry cassette (Figure 17).
Schematic of mCherry cassette chromosomal knock-in and location of specific primers for colony PCR screening.

Figure 17 – Schematic of mCherry cassette chromosomal knock-in and location of specific primers for colony PCR screening.


  • The resulting PCR products were run on an agarose gel to confirm correct chromosomal insertion (Figure 18).
Agarose gel depicting colony PCR screen for positive mCherry cassette integrants.

Figure 18 – Agarose gel depicting colony PCR screen for positive mCherry cassette integrants. An amplicon of 1400 bp is consistent with correct chromosomal integration. Lane 1: 1 kb Plus Opti-DNA Marker. Lane 2-6: Colonies #1-5.



b. Confirmation of mCherry Integration


  • Positive screened colonies were grown in liquid media and pelleted to reveal mCherry expression (Figure 19).
mCherry positive clones express red fluorescent protein thus producing a pink-red phenotype.

Figure 19 – mCherry positive clones express red fluorescent protein thus producing a pink-red phenotype. The wildtype E. coli strain is depicted in the first tube on the left.



c. Sequencing of mCherry Positive Colonies


  • PCR products were subjected to Sanger sequencing to confirm correct insertion and knock-in of the mCherry cassette (Figure 20).
CRISPR Cas9 - KO and KI Case Study 14

Figure 20 – Sequence alignment of mCherry positive colonies compared to wildtype and repair template sequences. The upstream sequence (green) confirms correct integration and a portion of the mCherry cassette sequence (red) is shown to differ from the wildtype sequence (blue).

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References
  • Kandarian SC, Nosacka RL, Delitto AE, Judge AR, Judge SM, Ganey JD, Moreira JD, Jackman RW. Tumour-derived leukaemia inhibitory factor is a major driver of cancer cachexia and morbidity in C26 tumour-bearing mice. J Cachexia Sarcopenia Muscle. 2018 Sep 30. doi: 10.1002/jcsm.12346.
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