A valuable way to study a gene's function and interactions is to compare the phenotype
of a wild-type organism with one in which the gene's activity has either been inactivated
or knocked-out.
In the past, knocking out a gene in an organism was very tedious, expensive, and time-consuming,
with few laboratories possessing the necessary resources to perform such experiments.
Thanks to the development of CRISPR technology, gene knockout experiments have now become
simple and accessible for almost any lab to perform.
In this CRISPR-based strategy, a Cas9 nuclease generates a double stranded break at a genomic
site specified by a single guide RNA.
The DNA break is then repaired via the cell's non-homologous end joining repair mechanism,
a mechanism that introduces site specific insertion/deletions (InDels) that can effectively
disrupt the gene's activity.
In this video, we will take you through a step-by-step case study of how CRISPR was
used to develop a biallelic LIF knockout in mouse colon carcinoma cells.
Our case study will also demonstrate how the knockout was verified using the Surveyor Assay
as well as Sanger and Next Generation Sequencing methods.
First, three sgRNAs were designed against the mouse LIF locus.
It is common practice to design more than one sgRNA to increase the likelihood of a
successful genomic edit.
Software analysis was performed to ensure the sgRNAs had no predicted off-target binding sites.
The selected sgRNA was then cloned into abm's all-in-one sgRNA and Cas9 lentivector.
In this lentivector, expression of the sgRNA is driven by the U6 promoter which is a strong
constitutive Pol III promoter.
To enable direct screening of the Cas9 expression via Puromycin selection, the Cas9 was incorporated
into a Cas9-2A-Puro cassette driven by an SFFV promoter.
This All-in-One lentivector was then packaged into a recombinant lentivirus using abm's
second generation lentiviral packaging system.
The recombinant lentiviruses were then transduced into mouse colon carcinoma cells at a multiplicity
of infection of 5.
After Puromycin selection, surviving cell colonies were isolated and their genomic DNA extracted.
A Surveyor assay was then performed to confirm whether genomic editing of the LIF locus has
taken place.
In this assay, the edited DNA strand is denatured and rehybridized with the wild-type strand
to create heteroduplexes that are mismatched at the mutation site.
The surveyor endonuclease cleaves the 3' end at both ends of mismatched sites, producing
cleavage products that are detectable via gel electrophoresis.
In our case, the surveyor assay produced two additional bands aside from the WT band from
colonies 3 and 6, indicating that a mismatch and consequently a successful edit had occurred.
On the other hand, colony 2 did not display additional bands, indicating there was no
successful editing.
To further characterize the nature of the knockout, colony 3 and 6 were analyzed via
Sanger Sequencing.
Sanger sequencing results showed that only one mutant sequence was detected in
colony 3.
This indicates that these cells are likely only heterozygotic knockouts.
On the other hand, colony 6 showed two different mutant sequences.
Further studies were performed on colony 6 to identify whether a frameshift mutation
had occurred.
A frameshift mutation disrupts the open reading frame, resulting in a nonsense mediated decay
of the mRNA transcript.
First, colony 6 was serial diluted into 96 well plates for monoclonal selection.
Genomic DNA was then extracted from these clones, PCR amplified, cloned, and sequenced.
Although clone 6a, 6b, and 6d all showed biallelic editing, sequencing results only showed a
biallelic frameshift mutation for clone 6a.
Further sequencing of clone 6a showed that only two mutant alleles were present, either
the 2 bp or the 4 bp deletion, and that no other WT or other mutations were detected.
Sanger Sequencing is a useful tool for obtaining sequencing information for up to a hundred
clones, however, this means a large proportion of the population is not accounted for.
For this reason, next generation sequencing methods can provide a significant advantage
as this method enables the sequencing of hundreds of thousands of alleles at one time, generating
a more complete dataset.
To evaluate knockout success, next generation sequencing was performed at each phase of
the project.
As expected, NGS results showed only WT sequences before editing was performed.
After Cas9 and sgRNA delivery, NGS results showed that the first round of selection resulted
in a mixture of 70% edited and 30% WT sequences in colony 6.
This indicates that there is a mixed population of edited and unedited cells, therefore, further
monoclonal selection is required.
Finally, after monoclonal selection, clone 6a showed 100% edited sequences, with no more
WT alleles remaining, indicating a fully validated and successful gene knockout.
Functional studies of LIF expression levels on the resulting cell line, indicated correct
CRISPR targeting in the cell line.
This data is courtesy of Dr. Robert Jackman from Boston University.
The CRISPR-based gene knockout technique is expanding the horizons for laboratories everywhere
in an unprecedented manner, enabling scientists to explore gene function and gene-protein
interactions like never before.
Backed by over a decade of experience in cell culture and cloning, our abm team of scientists
are equipped with the latest CRISPR tools and technologies to carry out any knockout
in any cell line you desire.
Our custom knockout cell line generation service offers sgRNA vector design, construction,
and delivery into your desired cell line, clonal screening and selection, and knockout
validation by Sanger sequencing.
Our CRISPR Knockout Services also includes a wide array of additional services, helping
you accomplish whatever project goal you have in mind.
Visit our website today to learn more about how we can help you with your gene editing
projects!