Non-genetically modified organism in vitro CRISPR/Cas9 gene editing of the lacZα gene: A 4.5 h laboratory course for senior high-school students
Abstract
The CRISPR/Cas9 system opens new horizons (M. Adli, Nat Commun, 2018) regarding genetic modifications of living organisms but also as an in vitro tool in laboratory protocols. Therefore, it boosts possibilities in research and future medical treatments. As the controversial claim of genomically edited babies by He Jiankui (Cyranoski D., Nature, 2019) demonstrates, the new gene editing potentials entail ethical discussions. A public or social discussion presupposes not only a theoretical knowledge or understanding of the system, but also profits from direct laboratory experiences showing how easy these techniques can be applied. Introducing numerous students and classes into these emerging techniques in a modern biology classroom depends on a suitable course concept, which fits legal and organizational requirements at the same time. Therefore, we implemented an appropriate hands-on laboratory course for senior high-school students, lasting just 4.5 h. Particularly with regard to European regulations concerning the handling of genetically modified organisms, the constructs and protocols avoid the transfer of Cas9 DNA. This normally mandatory transfer was replaced by in vitro gene-editing. This leads to Cas9 induced gene knock-outs due to frame shifts and/or the excision of DNA fragments in common Escherichia coli (E. coli) plasmids, such as pUC19. This gene knock-out concept covers various steps: In vitro plasmid editing with Cas9, ligation and transformation of E. coli cells with the modified plasmid DNA and finally the spread plating of transformed E. coli cells in order to analyze colonies after overnight incubation. The successful excision of DNA fragments by in vitro Cas9 treatment was determined by subsequent gel electrophoresis.
1 INTRODUCTION
This laboratory course for senior high-school students addresses various goals in the context of CRISPR/Cas9 function and application, modeling cellular processes and in vivo techniques with an in vitro system to demonstrate how easy new horizons1 regarding genetic modifications of living organisms can be opend up (leading to related ethical controversies2). First, a Cas9 mediated induction of a specific mutation resulting in a gene-knockout in a plasmid similar to actual research techniques3 is shown by the inactivation of the lacZα gene. For that purpose, one sgRNA target sequence is used, resulting in a single double-strand break. Second, the Cas9 cleavage mediated excision of a DNA fragment using CRISPR/Cas9 with two sgRNA target sequences4 is demonstrated by the deletion of the ampicillin resistance gene. Third, the visualization of Cas9 activity and its resulting effects is implemented by phenotype analysis and gel electrophoresis. The main focus of the course is the development of practical laboratory skills in the context of CRISPR/Cas9-mediated genome editing. Additionally, the protocol produces directly observable phenotypes and therefore supports the understanding of invisible subcellular processes which were already taught at school. The laboratory work involved can easily be accomplished in 4.5 h during a single course.
2 OVERVIEW
The CRISPR/Cas9 activity itself is limited to the highly specific induction of double-strand DNA breaks, guided or targeted by sgRNAs. The arising new gene editing potentials result from other cellular processes which occur in consequence to the DNA damage and are linked to different DNA repairing mechanisms. Here, we describe the concept and organization of a practical course where pUC19 DNA is used to demonstrate (a) the induction of double-strand breaks and the underlying specificity of this process by excision of a defined DNA fragment followed by gel electrophoresis and (b) a CRISPR/Cas9 induced gene-knockout or gene deletion as a consequence of a subsequent DNA repair. Figure 1 presents a schematic overview showing how this laboratory course is constructed in principle by illustrating the three work streams conducted in parallel, their timelines and experimental steps, tested with more than 300 students, working in teams of two.

2.1 Excision of a gene
To demonstrate the specific excision of a gene, two sgRNAs are used fitting the ampicillin resistance gene (ampR) of pUC19. An in vitro CRISPR/Cas9 assay is used to excise the fragment of 1052 bp. The fragment is detected by gel electrophoresis. By comparison with a DNA ladder the students can easily identify the length of the excised gene and the remaining pUC19 fragment. A detailed description of the sgRNAs used is given in Table 3, their corresponding positions on pUC19 are also shown in Figure 2.

2.2 CRISPR/Cas9 induced gene-Knockout
For the demonstration of a gene-knockout or gene editing approach a previously developed system,5 resulting in a detectable phenotype, was modified. Based on this concept a deletion of a few base pairs in pUC19 can be generated by in vitro CRISPR/Cas9 treatment followed by self-ligation. Guiding Cas9 with sgRNAs inducing a deletion of five bases directly after the start codon of the lacZα gene triggers a frameshift mutation in the gene. After transformation of Escherichia coli (E. coli) strains, which are suitable for blue-white selection, the frameshift mutation in lacZα can be detected by the occurrence of Ampicillin resistant clones, lacking the blue staining. By sequencing the plasmid DNA of such white clones, site-specific deletions/mutations can be identified and characterized.
2.3 Course structure and schedule
Published experimental procedures and protocols5 were modified to reduce the number of individual operations and the amount of time needed. By omitting uncritical experimental steps, the complexity of the experiment was reduced. This diminishes putative problems in understanding the general principles.
As shown in the schematic overview (Figure 1) all necessary steps, including both Cas9 treatments, gel electrophoresis, ligation, transformation and plating of E. coli cells can be completed in about 4 h and 30 min. After overnight incubation the grown colonies can be analyzed. Optionally the identification of induced DNA modifications can be done either by colony PCR or after plasmid isolation from selected and cultivated clones.
3 MATERIALS AND METHODS
3.1 Plasmids and strains
The pUC19 plasmid and competent E. coli NEB5α cells were obtained from New England Biolabs.
3.2 Chemicals, enzymes and kits
All Enzymes and kits for the various procedures were purchased from international suppliers to ensure reproducibility of the developed protocols and application without the need for further adjustments due to variances in stocks and their properties (Tables 1 and 2).
Chemicals for RNA protocols | |
RNAse away | Roth |
Nuclease free water | Roth |
In vitro CRISPR/Cas9 assay | |
SpCas9 Nuclease (1 μM) (Cas9, Streptococcus pyogenes) | New England Biolabs |
NEB3.1 buffer | New England Biolabs |
pUC19 (1 μg/μl) | New England Biolabs |
sgRNAs (100 μM) | Thermo-Fisher Scientific (Invitrogen) |
Proteinase K (800 Units/ml) | New England Biolabs |
RNAse A (10 mg/ml) | Thermo-Fisher Scientific |
Gel electrophoresis | |
Agarose MP BC | PanReac AppliChem |
10× buffer TBE (boric acid 0,89 M; EDTA-Na2 · 2H2O 0.02 M; Tris 0.89 M) | PanReac AppliChem |
Roti®-Load DNAstain 3 | Roth |
GeneRuler Express DNA ladder SM1551 | Thermo Fischer Scientific |
DNA purification | |
PCR clean-up gel extraction | Machery-Nagel |
Ligation | |
T4 DNA Ligase (400,000 Units/ml) | New England Biolabs |
10× T4 ligase buffer | New England Biolabs |
Transformation | |
Neb5®∝ competent Escherichia coli | New England Biolabs |
X-Gal (4% in DMSO) | PeqLab |
IPTG (100 mM) | Carl Roth |
LB-medium | |
Ampicillin | Sigma |
Plasmid-isolation | |
Plasmid DNA purification | Machery-Nagel |
DNA sequencing | |
Primer M13 fwd (100 μM) | Merck |
Primer CAP fwd (100 μM) | Merck |
Characterization of deletions inside of the lacZα gene | Binding position | |
---|---|---|
M13 fwd | 5’ GTAAAACGACGGCCAG 3‘ | inside the lacZα gene |
Proof of the excision of the lacZα gene | ||
---|---|---|
CAP fwd | 5′ TAGCTCACTCATTAGGCACC 3′ | Upstream of the lacZα gene |
Inactivation of lacZα gene | Result | |
---|---|---|
sgRNA lacZα | 5′ ATGCAAGCTTGGCGTAATCA 3′ | induction of base deletion |
excision of amp gene | ||
---|---|---|
sgRNA amp1 | 5′ ACGTCAGGTGGCACTTTTCG 3’ | amp fragment: 1052 bp |
sgRNA amp2 | 5′ AAAGTATATATGAGTAAACT 3’ | pUC19 fragment: 1634 bp |
3.3 Oligonucleotides for targeting (sgRNAs)
The sequences are given in Table 3. The target sites and corresponding sequences for gene excision are located at the start and end of the amp gene, the site for the induction of a mutation by a single double-strand break is located at the 5′ end of the lacZα gene (Figure 2).
4 EXPERIMENTAL PROCEDURES
4.1 Generation of double-strand breaks
A DNA sample of 30 μl containing 3 μl 60 nM pUC19 DNA is prepared as follows: 14 μl of nuclease-free water are pipetted into an Eppendorf-Tube before the following aliquots of necessary compounds are added: 3 μl 10× NEB3.1 buffer; 4 μl 1 μM SpCas9 nuclease; 3 μl 600 nM sgRNA amp1 and 3 μl 600 nM sgRNA amp2 (for fragment excision) or 6 μl 600 nM sgRNA lacZα (for gene knock-out). In one sample the volume of SpCas9 nuclease is replaced by nuclease-free water to get a negative control with undigested pUC19. The mixture is homogenized, briefly centrifuged and incubated at 25°C for 10 min. Subsequently 3 μl pUC19 DNA (60 nM, 0.316 μg plasmid) are added and the sample is incubated at 37°C in a heating block for 15 min.
In assays for fragment excision the reaction is stopped by adding 4 μl proteinase K and an incubation of 15–20 min at room temperature. The resulting samples are used for gel electrophoresis.
In assays performing gene knock-out the reaction is stopped by addition of 0.6 μl RNaseA and a 15 min incubation at 37°C in a heating block. Then 2.5 μl of proteinase K, 5 μl SDS solution 10%, 5 μl of 100 mM CaCl2 and 6.9 μl of nuclease-free water are added, followed by an incubation at 55°C for 30 min. The resulting samples are used for purification of the DNA.
4.2 Gel electrophoresis
The DNA samples (of the gene excision assay) are mixed with 6.8 μl of RotiLoad and then 20 μl of the samples are used in gel electrophoresis. 20 μl of DNA ladder are added into a separate slot as a length standard.
4.3 Purification of CRISPR/Cas9 treated pUC19 DNA
For DNA preparation the kit “PCR clean-up Gel Extraction” is used. The sample of approximately 50 μl is mixed with 125 μl of NTI buffer. For DNA binding, the complete batch is pipetted onto the column (NucleoSpin Gel and PCR Clean-up Column) and centrifuged at 11000 g for 30 s, the flow-through is discarded. The silica membrane is washed two times by adding of 700 μl of NT3 buffer onto the column and subsequent centrifugation for 30 s at 11000 g, discarding the flow-through. The DNA-loaded column is centrifuged again at 11000 g for 1 min to remove residual buffer. Finally, 15 μl of NE buffer are pipetted onto the column, which is placed in a 1.5 ml microcentrifuge tube. After incubation at RT for 1 min the column is centrifuged for 1 min at 11000 g, collecting the eluted DNA in the microcentrifuge tube.
4.4 Ligation
3 μl of the sample are mixed with 14 μl of nuclease-free water and then 2 μl of 10 × T4 DNA ligase buffer and 1 μl of T4 DNA ligase are added. This mixture is incubated at room temperature for 1 h. The reaction is stopped by incubation at 65°C for 10 min.
4.5 Transformation and phenotype analysis
The competent E. coli NEB5α cells are thawed on ice for 30 min. 50 μl of the cells are used for transformation. 10 μl of ligated DNA are added and the mixture is incubated on ice for 2 min. The cells are then heat-shocked at 42°C in a heating block for 30 s. The mixture is cooled down on ice for 2 min. After adding 200 μl of LB medium the mixture is incubated at 37°C on a laboratory shaker at 170 rpm for 30 min.
Subsequently 200 μl are spread-plated on agar plates (7%) containing ampicillin (200 μg/mL) with 50 μl X-Gal and 50 μl IPTG in the dark at 37°C for overnight incubation.
4.6 Plasmid isolation (optional for sequencing instead of performing colony PCR)
After blue-white screening, plasmid DNA from overnight cultures of selected white colonies is isolated by using the kit “Plasmid DNA Purification.” One white colony is transferred from the agar plate into a sterile test tube containing about 2 ml of LB-medium with ampicillin (0.2 mg/mL) using a sterile toothpick. After overnight incubation at 37°C and 170 rpm on a laboratory shaker the culture is transferred to one 1.5 ml reaction tube. This is centrifuged at 11000 g for 30 s. The supernatant is discarded. The cell pellet is resuspended in 250 μl of buffer A1 using a pipette. 250 μl of buffer A2 are added and the tube is inverted 6–8 times. This is followed by incubation at room temperature for 5 min. Subsequently 300 μl of buffer A3 are added. The tube is inverted until the blue discoloration completely disappears and then centrifuged at 11000 g for 5 min. The supernatant containing the DNA is loaded onto a column (nucleospin plasmid). After centrifugation of the column at 11000 g for 1 min, the DNA is bound to the matrix. Thereafter, 600 μl of buffer A4 are added for a washing step by centrifugation at 11000 g for 1 min. Subsequently the DNA-loaded column is placed back into the emptied reaction vessel and centrifuged at 11000 g for 2 min to dry the column. After addition of 50 μl of buffer AE onto the column it is incubated at RT for 1 min. By centrifugation at 11000 g for 1 min the DNA is eluted into a clean reaction vessel.
4.7 DNA sequencing (optional)
To prepare DNA samples for sequencing, the concentration of purified plasmid DNA isolated from batch-cultures of colonies with a white phenotype is measured using a spectrophotometer (NanoDrop). To generate DNA samples for the sequencing procedure plasmid aliquots of 10 μl with a concentration of 100 ng/μl are mixed with 2 μl primer (1 μM). Using this sample the sequencing procedure itself is performed by Seq-it GmbH & Co.KG. For the characterization of the mutation of the lacZα gene by a single CRISPR/Cas9 induced double-strand break, M13 fwd primers are used which allow sequencing of the plasmid region where an elicited deletion was expected.
5 RESULTS
5.1 Gene excision
Analysis by gel electrophoresis shows in the negative control (Figure 3a, Lane C) as expected one fragment which corresponds to an untreated pUC19. In comparison with the DNA ladder the fragment shows a length of approximately 1500 bp. Treatment with CRISPR/Cas9 nuclease results in three fragments (Figure 3a, Lane T), with the length of approximately 2686 bp, 1634 bp and 1052 bp. These lengths correspond well to our expected lengths. The two shorter fragments are expected when both targeted sites are cut by CRISPR/Cas9. It is visible that the in vitro CRISPR/Cas9 treatment induces doublestrand breaks. The longest fragment is the result of only one target site being cut by CRISPR/Cas9, resulting in a linearized pUC19 vector. This pattern is observed using 1% agarose gels and TAE-buffer followed by detection with Evagreen. Between 85% and 94% of the samples prepared by students showed the expected results.

5.2 CRISPR/Cas9 induced mutation of the lacZα gene
Induction of a single double-strand break in pUC19 with CRISPR/Cas9 and sgRNA lacZα followed by ligation and subsequent transformation of competent NEB5α cells generated white colonies on LB agar plates containing ampicillin (Figure 3c). Just blue colonies were observed after plating of NEB5α cells transformed with untreated fractions of pUC19 (Figure 3d). Figure 3b shows the number of white and blue colonies obtained. Sequencing of white colonies showed, that the CRISPR/Cas9 treatment and the subsequent ligation led to a deletion of five nucleotides in the lacZα gene (Figure 4). More than 82% of the spread plates prepared by students for phenotype monitoring show the expected phenotypes and colony type distribution, indicating that students are able to perform the laboratory tasks and analyses successfully.

6 DISCUSSION
The results show that with the protocols described an in vitro gene editing and knock-out is feasible in just 4.5 h. By an optional sequencing step highly reproducible deletions leading to the knock-out phenotype can be analyzed and demonstrated on the nucleotide level. According to the obtained data the two parts of this laboratory course yielded promising results for teaching CRISPR/Cas9 related biological content and knowledge.
The appearance of three bands (Figure 3a, Lane T) as result of the applied CRISPR/Cas9 treatment can be interpreted and discussed – also by the students – as follows:
The CRISPR/Cas9 endonuclease activity results in DNA double-strand breaks (dsDNA). When only one target site is cut, the vector is linearized. Since two distant sites are targeted to be cut with two different CRISPR/Cas9–sgRNA complexes, the linearization could be the result of a dsDNA break at either site, yielding a band of 2686 bp (Figure 3a, Lane T), no matter at which of the targeted positions the dsDNA break is induced successfully. A possible modification of the concept to stress this insight would be the integration of two additional assays containing just one of the sgRNA polymers. The induction of two dsDNA breaks results in the formation of two fragments differing in length, one fragment containing the ampicillin resistance gene (1052 bp) and the other one the remaining part of the pUC19 vector (1634 bp). Therefore, the three bands shown in Lane T indicate successful excision of the ampicillin fragment by CRISPR/Cas9, as intended.
The control lane (Figure 3a, Lane C) shows the supercoiled pUC19 vector. The apparent fragment size of the band corresponds to 1500 bp, which is significantly smaller than the linearized vector in the treatment lane. This is expected, as it is known that the migration behavior of supercoiled vectors is similar to smaller fragments.
Regarding the proportion of correct results this part of the laboratory course and the corresponding protocols were uncritical and unproblematic when carried out by students, which is mainly because it is quite short and simple. In almost all cases there was successful excision of the ampicillin resistance gene visible after gel-electrophoresis. Unexpected results, such as missing DNA bands in the gel, were mostly due to faulty application of the samples while loading the DNA into the gel. Therefore, it is vital to demonstrate students how to properly apply their samples onto the gel in order to obtain best results. This part of the protocol for the demonstration of the Cas9 activity and the DNA modification shows excellent reproducibility.
In the second part of the course white colonies are expected as a result of the mutagenesis of the lacZα gene (Figure 3c). The ratio of white colonies to blue colonies indicates the efficiency of the Cas9 nuclease. The mutation in the lacZα gene is induced by targeting a single CRISPR/Cas9 site, at the 5′ end of the lacZα gene in pUC19 (Figure 2). Since untransformed NEB5α cells do not carry a genomic ampicillin resistance, all observed colonies must contain and express pUC19 DNA. Transformed NEB5α cells can survive on plates containing ampicillin but are – in case of a mutated lacZα gene – not able to express a functional ß-Galactosidase enzyme by alpha complementation after induction with IPTG. Therefore X-Gal is not hydrolyzed and no coloring occurs.
All blue colonies are formed by NEB5α cells that were transformed with pUC19 in which no dsDNA break occurred. This is expected since CRISPR/Cas9 is not always successful in inducing a break of the dsDNA, which also leads to just linearized vector in Lane T (Figure 3a). Another explanation would be a mutagenesis resulting in the deletion of a DNA triplet, leading to a functional ß-Galactosidase. Theoretically also a DNA repair after transformation with a linearized vector could result in a functional lacZα gene. However, this is highly unlikely, as the transformation rate of linearized DNA is very low.6 Moreover, it is known that cutting DNA with CRISPR/Cas9 can result in deletion of a few bases, as can be seen in our sequencing data (Figure 4). The number of blue colonies was higher than the number of white colonies (Figure 3b). In our experience the ratio of white to blue colonies varies much between experiments. A low ratio resulting in only a few white colonies can occur due to incomplete cleavage by CRISPR/Cas9 as discussed above and/or incomplete ligation of linearized and mutagenized vector.
Moreover, religation of cut pUC19 results in circular DNA, which is not supercoiled (since no Topoisomerase is present). The transformation efficiency using religated pUC19 is lower compared to uncut, supercoiled pUC19,7 which biases for blue colonies.
The data in Figure 4 show the sequencing results of 10 different white colonies, picked from various sample plates. All clones show the same deletion, indicating that five base pairs have been removed by an exonuclease activity of CRISPR/Cas9 under the conditions applied, resulting in a frame shift mutation. This leads to a loss of function of the lacZa gene, resulting in the formation white colonies as detected on the plates (Figure 3c).
The vast majority of students can produce the expected results. In comparison with other student courses conducted in our labs, the rate of unexpected results is not unreasonably high, showing that the concept can be successfully applied by students. However, the most frequent problems observed are plates without white colonies, plates with no colonies at all or plates with too many colonies and additional satellite colonies. One of the likely reasons for these failures is the pipetting of small quantities, like 1 μl ligase. To successfully add the correct amount, precision when using the pipette is needed. The 30 s heat shock proved to be one of the critical tasks, mainly because it showed to be necessary to time it correctly and put the samples back into the ice bath straightaway, which sometimes students forgot. This could result in cell death or lower the transformation rate, therefore reducing the chances for good results. Other reasons are the numerous steps of the protocols, which increase the probability of making a mistake.
The project requires different levels of assistance by the supervisor, depending on the skill level of a class. Yet, we could establish that there was enough time between experimental steps to cover the theoretical background of CRISPR/Cas9 and to explain upcoming steps/basic laboratory work. This helps students to get a better understanding of the concept and reduces the number of mistakes. We highly suggest using available time slots (e.g., during incubation) to talk about the concept and upcoming steps – here recapitulating certain important parts several times can be beneficial – to further strengthen better understanding of the required steps. However, based on our experience the results of the mutagenesis part of the course showed greater variability than the one of the fragment excision experiment.
It should also be mentioned that the results of the fragment excision experiment can be obtained directly at the end of the course (gel electrophoresis), while the results of the mutagenesis experiment require over-night incubation of the plated cells. In our concept, pictures of the plates are provided the following day.
ACKNOWLEDGMENTS
Open Access funding enabled and organized by Projekt DEAL.