How to perform a CRISPR Knockout Experiment

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!


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