How to Edit Genes (Using CRISPR)

Keith Jacobs received his Ph.D. in Molecular Cell Biology from Washington University in St. Louis in 2015. After a short year of postdoctoral research, Keith will soon be working as a Biologist for the Environmental Protection Agency. Check out his personal blog at http://blabberingbiologist.blogspot.com , and twitter feed @blabberbiology.

​Clustered regularly interspaced short palindromic repeats (CRISPR) technology is revolutionizing molecular biology and genetic engineering. The CRISPR system allows selective permanent editing of almost any genomic region, whether by deletion, insertion or substitution of specific nucleotides (units of DNA). CRISPR utilizes a short RNA molecule (similar to DNA, but used by cells to put genes in their active form) that is complementary to the genomic region of interest, which targets the DNA for cleavage by the Cas9 protein. CRISPR/Cas9 therefore has almost unlimited potential, not just for laboratory research studies but more importantly for potentially curing genetic diseases.
 
CRISPR is a naturally occurring system of acquired immunity (aka an immune system) in many species of bacteria that protects against viral infection. CRISPR regions in bacteria, first discovered in 1987, contain a large chain of nucleotide repeats separated by unique short sequences called spacers along with a series of associated genes (called cas) at one end of the chain. Several studies in the mid to late 2000s later determined that spacer sequences matched genomic regions from specific viruses and were associated with resistance to those viruses. CRISPR therefore acts like an ancient bacterial form of the antibody-mediated immunity (the mechanism by which our body defends against past infection) that is present in mammals.
 
CRISPR-mediated immunity works in three phases. First, a new spacer – a piece of DNA obtained from an invading virus – must be integrated into a bacterium. Next, the CRISPR region – the chain of repeats – is expressed (“read”) and individual spacer sequences are processed into what are called crRNAs (CRISPR RNAs). crRNAs can then recognize the complementary sequence in an invading virus, targeting its genome for destruction. Viral DNA sequences are selected for integration by 2 members of the cas family, Cas1 and Cas2, which recognize short sequences known as protospacer-adjacent motifs (PAM sequences). The presence of a PAM sequence is required for Cas binding, but they are broadly distributed throughout the genome. Cas1 and Cas2 cut the viral DNA adjacent to the PAM sequence and insert that region into one end of the CRISPR array. The total array is expressed as a single long RNA, and groups of Cas proteins then process this RNA into individual crRNAs containing each individual spacer sequence. These sequences, which were derived from the genome of invading viruses and are therefore complementary to their DNA, are able to target the genome of future invading viruses. Upon future infection, crRNAs recruit either a single or series of Cas proteins to the targeted location in the viral genome, leading to cleavage by Cas proteins and DNA degradation. In order to prevent the system from accidentally destroying the host genome where the CRISPR array is integrated, crRNAs retain part of the repeat non-spacer sequence at each end. This helps them discriminate between their complementary foreign DNA (which does not contain the repeat sequence) and the host sequence.
 
The mechanism of CRISPR interference is analogous to RNA interference (RNAi) in eukaryotes (more advanced cells than bacteria). In RNAi, short pieces of RNAs are also processed from longer precursors in order to target effector proteins (similar to crRNAs recruiting Cas) toward complementary gene sequences in order to inhibit their function. However, unlike crRNAs, microRNA (miRNA) and short interfering RNA (siRNA) as part of RNAi target RNA instead of genomic DNA. Additionally, miRNAs and siRNAs are processed differently. Short interfering RNA (siRNA) are not naturally occurring but can be derived from an invading piece of DNA similar to crRNAs, and target either their own mRNA (expressed genes) or invading RNA viruses for degradation. miRNA in contrast are naturally expressed and are responsible for fine tuning of the cell’s own gene expression. RNAi is commonly used in molecular biology research for easily downregulating gene expression temporarily, in contrast with CRISPR which creates permanent genomic changes.
 
Molecular biologists have been taking advantage of CRISPR function in order to create targeted permanent gene mutations. Guide RNAs, essentially manufactured spacer sequences, can be designed to target any genomic location with an adjacent PAM sequence. Co-expressed Cas9 protein is then recruited to the site marked by the guide RNA where it induces a DNA break. As the cell attempts to repair the break through natural mechanisms, errors during repair result in a variety of mutations that can inactivate the gene. Alternatively, a repair template may be provided along with the guide RNA and Cas9 protein in order to incorporate site-specific mutations. As Cas9 induces DNA breaks, the cell will attempt to use the provided template for error-free repair. Any nucleotide-specific modifications present in the template will therefore be incorporated into the genome, replacing the original sequence.
 
CRISPR is not the first system created for site-specific genome modification, however its low cost, greater efficiency, and simplicity make it revolutionary in its potential applications. Researchers can use CRISPR to study mutations not only of whole genes but of individual nucleotides and amino acids (protein building blocks), while physicians could potentially use the system to cure genetic diseases or add genetic functions to particular cell types. While CRISPR’s potential may be endless, the technology does still require some improvements before it can be relied on for clinical use. Despite the specificity of guide RNAs, off-target effects are often seen, although slight modifications to the system have already been successful in reducing these issues. More importantly, delivering a CRISPR vector to only the desired tissue type while avoiding toxicity may prove especially challenging. Despite these issues, the world’s first clinical trial involving CRISPR gene editing has recently been approved. In this trial, immune cells of the blood are isolated and removed, edited, and subsequently returned into circulation in order to target cancer cells for destruction. Blood cells are much easier to modify than internal organs and other tissues, since they can be genetically edited outside of the body. If this trial goes well, CRISPR-based genetic engineering may soon become a commonly-used tool for curative medicine.

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