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Learn how the CRISPR/Cas9 system works for gene-editing, how it can effectively create knock-out (KO) cell lines, and the advantages & considerations of this technique.
The ability to determine the entire sequence of the human genome has enabled researchers to identify disease-specific mutations and explore the basis of many genetic diseases. It has also led to the desire to be able to remove and modify particular genes to study their function.
Examining how biochemical pathways function or dysfunction in the absence of a specific protein can provide insight into the role that the protein normally plays. It may provide a direct functional contribution or form part of a regulatory feedback process. In addition, the ability to knock-out specific genes with a high level of accuracy facilitates the development of improved models of diseases with a gene-based etiology, which in turn can inform new treatments and novel genetic therapies.
Genetic sequences were initially modified using a range of DNA-cleaving techniques, such as zinc-finger nucleases (ZFNs) and TAL effector nucleases (TALENs). However, these were complex and time-consuming. The subsequent discovery of CRISPR/Cas9 genome editing technology has enabled genes to be edited with greatly improved specificity. This has further extended the potential for manipulating DNA1.
Clustered regularly interspaced short palindromic repeats (CRISPR), is the the DNA that makes up the bacterial adaptive immune system. It is used to help protect bacteria against invading viruses and facilitate their destruction by providing a memory of target DNA sequences to cut. These repeating spacer sequences guide nucleases to cut DNA of the same sequence that invades the cell in the future2.
This DNA cleavage is achieved by the enzyme Cas9, which has the ability to cut through DNA. Composed of single-stranded RNA, transcribed CRISPR sequences act as a guide for the enzyme enabling it to disable and disrupt the viral genome at very specific locations.
The molecular components of the CRISPR/Cas9 system are now used as a gene-editing system in eukaryotic cells1. CRISPR/Cas9 gene-editing uses short synthetic stretches of guide RNAs to direct the Cas9 enzyme to cleave a target location in the genome. The enzyme Cas9 is directed to the target where it cleaves the DNA three bases from the end of the target sequence.
This CRISPR/Cas9 system provides a robust means to allow precise genomic sequences to be replaced, deleted or inserted. The pattern of Cas9 activity is determined by the nucleotides that surround the target cleavage site and a set of rules has been compiled describing these effects so CRISPR-Cas9 DNA modifications can be reliably and precisely predicted3.
A whole gene can be excised to study its function or precise point mutations inserted to replicate those known to cause disease in human patients to provide models of gene-based disorders.
CRISPR/Cas9 has brought a new level of accuracy and specificity to gene editing that has made it possible to conduct experiments that were previously impossible. It is three to four times more efficient than the traditional ZFN and TALEN systems4. Furthermore, multiple genes can be deleted simply by introducing several different guide RNAs5.
The technique has been widely adopted for the creation of knock-out cell lines, in which the target gene can be removed with precision, resulting in complete ablation of the protein it encodes. This enables gene function and the interplay between genes to be studied and facilitates in-depth research into human genetic disorders.
There are various considerations that should be taken into account to optimize the precision and success of CRISPR/Cas9 knock-out.
A common problem with all genome editing methods is off-target effects that arise when a nuclease cleaves DNA in a place other than where it was intended. Such unplanned DNA breaks could give rise to additional effects not related to the target gene. Consequently, a suitable single nucleotide polymorphism or fluorescent tag can be incorporated to verify the accuracy of the knock-out. Such verification steps require special care to avoid misinterpreting naturally occurring polymorphisms as off-target mutations.
Once the knock-out has been undertaken, it is necessary to validate that the desired gene has been removed. A phenotype can be used as an initial indicator of success. However, molecular techniques will usually also be used to definitively characterize the editing event. As a first pass, DNA mismatch detection assays can verify the CRISPR-Cas9 reaction resulted in knock-out, but full sequencing of the DNA is essential to ultimately verify knock-out of all alleles in a clonal cell line, without any unwanted additional deletions.
CRISPR knockouts provide a great opportunity to test gene function, interplay, and develop disease models to research potential treatments. However, developing the knockouts requires expertise and can be time-consuming, particularly if a laboratory does not already have the required technical skillset.
To enable researchers to take advantage of the benefits of knock-out cell lines without having to acquire expertise in CRISPR-Cas9 gene-editing techniques, it is possible to purchase a wide variety of CRISPR-Cas9 knock-out cell lines or lysates.
Abcam offers an extensive range of knock-out cell lines and lysates suitable for a wide range of research applications6. Indeed, Abcam hosts the industry’s largest library of immortalized diploid knock-out cell lines, including Hela, HEK293T, A549, HCT116, Hep G2, and MCF.
Each knockout cell line and lysate is generated using standardized CRISPR-Cas9 methodology and accompanied by the parental wild-type lysate to allow the biological impact of the knockout to be assessed within a consistent cellular background. The cell lines are individually cloned and validated by Sanger sequencing and often include western blot, to ensure the accuracy of the edit.