The CRISPR genome-editing technology is grabbing significant headlines in the biomedical world. In less than three years, the number of articles published on this technology has gone from only a few to over a dozen per week. CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, and its associated protein, Cas9, provide sequence-specific adaptive immunity in bacteria and archaea by integrating short viral DNA sequences in the host cell genome, allowing the cell to remember, recognize, and clear infections (1). The most attractive feature of CRISPR-Cas9 is that it offers a more simple and cost-effective way to delete, insert, or replace genes, as compared to other gene editing methods such as those based on TALENs (transcription activator-like nucleases) or ZFNs (zinc-finger nucleases). The range of applications using CRISPR-Cas9 is broad, and scientists have taken advantage of this system to edit the genomes of human cells and correct genetic diseases in mice. These recent applications provide the proof-of-principle that gene therapy using CRISPR-Cas9 may be possible in patients in the future. However, more research needs to be done to translate this technology into a safe and reliable therapy that can be used to treat genetic diseases.
What is CRISPR?
The CRISPR-Cas system was first discovered in E. coli in 1987 (2). Its function was later established in a species of bacteria, S. thermophilus, by showing that CRISPR provides acquired resistance against infectious viruses (3). There are three types of CRISPR-Cas systems, but the type II system utilized by the bacteria Streptococcus pyogenes is the most commonly used and extensively characterized system to date. In the type II system, invading DNA from viruses is cut up and incorporated into the bacterial genome at the CRISPR loci amidst a series of short repeats (4,5). The CRISPR loci are then transcribed, and the transcripts are processed to generate small RNAs named crRNA, which target the Cas9 endonuclease to the foreign DNA based on sequence complementarity. Specifically, Cas9 endonuclease participates in site-specific recognition and cleavage of target DNA by forming a complex with both crRNA and a separate trans-activating crRNA (tracrRNA) that is partially complementary to the crRNA (6). (Fig. 1: mechanism). Scientists have successfully combined crRNA and tracrRNAinto a single synthetic guide RNA (sgRNA) for targeted gene-editing purposes in animal and plant cells (7). Together with a plasmid that expresses the Cas9 endonuclease, this combination represents a simple two-component system that can be adapted to any genomic sequence simply by changing the sgRNA sequence. Additionally, sgRNAs can be multiplexed to generate numerous genetic alterations simultaneously.
CRISPR-Cas9 has revolutionized the gene editing field because it is an efficient new way to induce sequence-specific genome editing in a variety of cell types and organisms that have traditionally been challenging to manipulate genetically. Cas9 has been employed to induce deletions or inversions of genes in human cancer cells (8,9), skin cells (10), embryonic stem cell clones (11), and in a wide variety of model organisms (12). Aside from its genome-editing feature, the CRISPR-Cas9 system can be used to regulate endogenous gene expression in living cells or organisms. A catalytically inactive Cas9 that lacks endonuclease activity has been fused to transcriptional activator or repressor domains and was shown to regulate the expression of endogenous genes (13,14). CRISPR-Cas9 can also be used to study chromosome dynamics and structure in living cells. For example, imaging repetitive elements in telomeres has been achieved using a GFP-tagged endonuclease deficient Cas9 protein and a structurally optimized sgRNA (15).
Importantly, CRISPR-Cas9-mediated genome manipulations in human cells and mouse models have demonstrated the therapeutic potential of this gene-editing tool for a variety of ailments. Cas9-mediated screens have identified genes responsible for cell survival and drug resistance in various cancer cells (16, 17). An in vivo genome-wide CRISPR-Cas9 screen has been used to identify the genes regulating lung metastasis (18). The CRISPR-Cas9 system has also been applied as a strategy to fight infections. Cas9 has been developed as an antimicrobial agent to specifically target antibiotic-resistant bacteria (19) and has shown therapeutic potential against HIV by suppressing viral gene expression and replication (20). Furthermore, CRISPR-Cas9-mediated gene therapy has been applied to stem cells obtained from cystic fibrosis patients to repair a deletion in the transmembrane conductor receptor gene that causes the protein to misfold (21), and it has been used in vivo to correct the muscular dystrophy-associated dystrophin gene mutation in the germ line of mice. Finally, researchers have generated cataract-free mice by injecting mutant zygotes with CRISPR-Cas9 to replace a single base pair mutation that causes cataracts (22).
However, there are many obstacles to realizing the full therapeutic potential of CRISPR-Cas9. One limitation of this gene editing approach is that it could create off-target effects by genetically altering sites other than the target DNA (23). There is also a lack of understanding regarding how epigenetics affect cleavage by Cas9. Although one study suggests that DNA methylation does not inhibit Cas9-based gene editing (24), further studies are required to determine whether chromatin structure has an impact on Cas9 cleavage or sgRNA targeting. Conversely, the consequences of altering the epigenetic state of the genome by CRISPR-Cas9 technology should also be investigated.
To increase the gene-editing specificity of CRISPR-Cas9 for potential therapeutic applications, more strategies need to be developed to reduce non-homologous end joining (NHEJ) alterations and increase homology directed repair (HDR). These two competing repair pathways can lead to either gene correction or gene inactivation and must be tightly controlled. For instance, HDR-induced altered gene sequences in the protospacer region of the target site could be additionally re-cut and subjected to mutagenesis by NHEJ, thereby reducing the yield of correctly edited alleles (25). According to Jennifer Doudna of U.C. Berkeley, one of the pioneers of CRISPR-Cas9 technology, on-target mutagenesis by NHEJ could have confounding effects where during the process of editing a deleterious mutation, “one could unintentionally convert sickle cell disease into beta thalassemia” (26). Feng Zhang’s group at MIT has already taken steps to increase the precision of the Cas9 system by developing a mutant Cas9 protein (Cas9D10A) that does not activate NHEJ (9). Rather, when provided with a homologous repair template, Cas9D10A repairs DNA exclusively through the HDR pathway, reducing unintended insertions and deletions.
CRISPR-Cas9 has gone through an explosive period of growth from its discovery as an innate immune function in prokaryotes against foreign DNA to its current use in editing genes in various cells and organisms. As researchers move forward with the CRISPR-Cas9 gene editing system, it is important to keep in mind that the biological consequences of altering genes in a cell or organism through this technology could be difficult to predict. In a recent interview in the journal Nature Biotechnology (26), Feng Zhang said, “Technologically, we don’t know how specific the current generation of genome-editing tools is. Do these tools result in any other changes in the genome? Do they affect the cell in other undesirable ways, such as altering the epigenetic state of the genome and lead to other lasting consequences?” These questions indicate that much more research needs to be performed to determine the safety and efficacy of CRISPR-Cas9 as a potential tool for gene therapy.
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