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An Overview of the CRISPR Cas9 Genome Editing System


An Overview of the CRISPR Cas9 Genome Editing System


The clustered regularly interspaced short palindromic repeats (CRISPR) associated sequences (Cas) system is a prokaryotic acquired immunity against viral and plasmid invasion. The CRISPR Cas9 system is highly conserved throughout bacteria and archaea. Recently, CRISPR/Cas has been utilized to edit endogenous genomes in eukaryotic species. In certain contexts, it has proven invaluable for in vitro and in vivo modeling. Currently, CRISPR genome editing boasts unparalleled efficiency, specificity, and cost compared to other genome editing tools, including transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs). This review discusses the background theory of CRISPR and reports novel approaches to genome editing with the CRISPR system.


CRISPR as a prokaryotic adaptive immune system

CRISPR was originally discovered in bacteria1 and is now known to be present in many other prokaryotic species.2,3 CRISPR systems in bacteria have been categorized into three types, with Type II as the most widely found. The essential components of a Type II CRISPR System located within a bacterial genome include the CRISPR array and a Cas9 nuclease. A third component of the Type II system is the protospacer adjacent motif on the target/foreign DNA. The CRISPR array is composed of clusters of short DNA repeats interspaced with DNA spacer sequences.4 These spacer sequences are the remnants of foreign genetic material from previous invaders and are utilized to identify future invaders. Upon foreign invasion, the spacer sequences are transcribed into pre-crisprRNAs (pre-crRNAs), which are further processed into mature crRNAs. These crRNAs, usually 20 base pairs in length, play a crucial role in the specificity of CRISPR/Cas. Upstream of the CRISPR array in the bacterial genome is the gene coding for transactivating crisprRNA (tracrRNA). tracrRNA provides two essential functions: binding to mature crRNA and providing structural stability as a scaffold within the Cas9 enzyme.5

Post-transcriptional processing allows the tracrRNA and crRNA to fuse together and become embedded within the Cas9 enzyme. Cas9 is a nuclease with two active sites that each cleaves one strand of DNA on its phosphodiester backbone. The embedded crRNA allows Cas9 to recognize and bind to specific protospacer target sequences in foreign DNA from viral infections or horizontal gene transfers. The crRNA and the complement of the protospacer are brought together through Watson-Crick base pairing. Before the Cas9 nuclease cleaves the foreign double-stranded DNA (dsDNA), it must recognize a protospacer adjacent motif (PAM), a trinucleotide sequence. The PAM sequence is usually in the form 5’-NGG-3’ (where N is any nucleotide) and is located directly upstream of the protospacer but not within it. Once the PAM trinucleotide is recognized, Cas9 creates a double-stranded breakage three nucleotides downstream of the PAM in the foreign DNA. The cleaved foreign DNA will not be transcribed properly and will eventually be degraded.5 By evolving to target and degrade a range of foreign DNA and RNA with CRISPR/Cas, bacteria have provided themselves with a remarkably broad immune defense.6

CRISPR Cas9 as an RNA-guided genome editing tool

The prokaryotic CRISPR/Cas9 system has been reconstituted in eukaryotic systems to create new possibilities for the editing of endogenous genomes. To achieve this seminal transition, virally-derived spacer sequences in bacterial CRISPR arrays are replaced with 20 base pair sequences identical to targeting sequences in eukaryotic genomes. These spacer sgRNAsequences are transcribed into guide RNA (gRNA), which functions analogously to crRNA by targeting specific eukaryotic DNA sequences of interest. The DNA coding for the tracrRNA is still found upstream of the CRISPR array. The gRNA and tracrRNA are fused together to form a single guide RNA (sgRNA) by adding a hairpin loop to their duplexing site. The complex is then inserted into the Cas9 nuclease. Within Cas9, the tracrRNA (3’ end of sgRNA) serves as a scaffold while the gRNA (5’ end of sgRNA) functions in targeting the eukaryotic DNA sequence by Watson-Crick base pairing with the complement of the protospacer (Fig. 1). As in bacterial CRISPR/Cas systems, a PAM sequence located immediately upstream of the protospacer must be recognized by the CRISPR/Cas9 complex before double-stranded cleavage occurs.5,7 Once the sequence is recognized, the Cas9 nuclease creates a double-stranded break three nucleotides downstream to the PAM’s location in the Eukaryotic DNA of interest (Fig. 1). The PAM is the main restriction on the targeting space of Cas9. Since the PAM is required to be immediately upstream of the protospacer, it is theoretically possible to replace the 20 base pair gRNA in order to target other DNA sequences near the PAM.5,7

Once the DNA is cut, the cell's repair mechanisms are leveraged to knockdown a gene, or insert a new oligonucleotide into the newly formed gap. The two main pathways of double-stranded DNA lesion repair associated with CRISPR genome editing are non-homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ is mainly involved with gene silencing. It introduces a large number of insertion/deletion mutations, which manifest as premature stop codons, that effectively silence the gene of interest. HDR is mainly used for gene editing. By providing a DNA template in the form of a plasmid or a single-stranded oligonucleotide (ssODN), HDR can easily introduce desired mutations in the cleaved DNA.5

The beauty of the CRISPR system is its simplicity. It is comprised of a single effector nuclease and a duplex of RNA. The endogenous eukaryotic DNA can be targeted as long as it is in proximity to a PAM. The goal of this system is to induce a mutation, and the CRISPR Cas9 complex will cut at the site repeatedly until a mutation occurs. When a mutation does occur, the site will no longer be recognized by the complex and cleavage will cease.

Optimization and specificity of CRISPR/Cas systems

If CRISPR systems are to be widely adopted in research or clinical applications, concerns regarding off-target effects must be addressed. On average, this system has a target every eight bases in the human genome. Thus, virtually every gRNA has the potential for unwanted off-target activity. Current research emphasizes techniques to improve specificity, including crRNA modification, transfection optimization, and a Cas9 nickase mutation.

The gRNA can be modified to minimize its off-target effects while preserving its ability to target sequences of interest. Unspecific gRNA can be optimized by inserting single-base substitutions that enhance its ability to bind to target sequences in a position and base-dependent manner. Libraries of mutated genes containing all possible base substitutions along the gRNA have been generated to examine the specificity of gRNA and enzymatic activity of Cas9. It is important to note that if mutations occur near the PAM, Cas9 nucleases do not initiate cleavage. Targeting specificity and enzymatic activity are not affected as strongly by base substitutions on the 5’ end of gRNA. This leads to the conclusion that the main contribution to specificity is found within the first ten bases after the PAM on the 3’ end of gRNA.5

The apparent differential specificity of the Cas9 gRNA guide sequence can be quantified by an open source online tool ( This tool identifies all possible gRNA segments that target a particular DNA sequence. Using a data-driven algorithm, the program scores each viable gRNA segment depending on its predicted specificity in relation to the genome of interest.

Depending on the redundancy of the DNA target sequence, scoring and mutating gRNA might not provide sufficient reduction of off-target activity. Increasing concentrations of CRISPR plasmids upon transfection can provide a modest five to seven fold increase in on-target activity, but a much more specific system is desirable for most research and clinical applications. Transforming Cas9 from a nuclease to a nickase enzyme yields the desired specificity.5 Cas9 has two catalytic domains, each of which nicks a DNA strand. By inactivating one of those domains via a D10A mutation, Cas9 is changed from a nuclease to a nickase.

Two Cas9 nickases (and their respective gRNAs) are required to nick complementary DNA strands simultaneously. This technique, called multiplexing, mimics a double-stranded break by inducing single-stranded breaks in close proximity to one another. Since single-stranded breaks are repaired with a higher fidelity than double-stranded breaks, off-target effects caused by improper cleavage can be mitigated, leaving the majority of breaks at the sequence of interest. The two nickases should be offset 10—30 base pairs from each other.5 Multiplex nicking offers on-target modifications comparable to the wild type Cas9, while dramatically reducing off-target modifications (1000—1500 fold).5


CRISPR/Cas9 systems have emerged as the newest genome engineering tool and have quickly been applied in in vitro and in vivo research applications. However, before these systems can be used in clinical applications, off-target effects must be controlled. In spite of its current shortcomings, CRISPR has proven invaluable to researchers conducting high-throughput studies of the biological function and relevance of specific genes. CRISPR Cas9 genome editing provides a rapid procedure for the functional study of mutations of interest in vitro and in vivo. Tumor suppressor genes can be knocked out, and oncogenes with specific mutations can be created via NHEJ and HDR, respectively. The novel cell lines and mouse models that have been created by CRISPR technologies have thus far galvanized translational research by enabling more perspectives of studying the genetic foundation of diseases.


  1. Ishino, Y. et al. J Bacteriol. 1987, 169, 5429–5433.
  2. Mojica, F.J. et al. Mol Microbiol. 1995, 17, 85–93.
  3. Masepohl, B. et al. Biophys Acta. 1996, 1307, 26–30.
  4. Mojica, F.J. et al. Mol Microbiol. 2000, 36, 244–246.
  5. Cong, L. et al. Science. 2013, 6121, 819–823.
  6. Horvath, P. et al. Science. 2010, 327, 167.
  7. Ran, F.A. et al. Nat. Protoc. 2013, 8, 2281–2308.


A Cup of Tea Against Cancer


A Cup of Tea Against Cancer

Green tea, made from the leaves of Camellia sinensis, has come a long way from its humble origins in China to its current status as the second most popular beverage worldwide. According to Chinese mythology, Shennong, the legendary ruler of China in approximately 2370 BC, drank the first cup of green tea that was brewed when a tea leaf fell into his boiled water.1 Despite his title as the divine healer, Shennong could not have possibly realized the numerous health benefits contained in the little cup. Green tea benefits health in various ways including cognitive enhancement, improvement of mental ability and alertness,2 and increased reward learning through modulation of dopamine transmission.3 Tea also helps with dieting through increased fat oxidation and prevents cardiovascular disease and diabetes.4 Recently, several studies have also credited green tea for its ability to prevent cancer development.1,4-6

When harvested from the tree, leaves of Camellia sinensis contain a high concentration of flavonoids. Flavonoids are members of the polyphenol group and have demonstrated anti-inflammatory, anti-allergic, and anti-mutagenic effects. In green tea, a group called catechin constitutes a large percentage of the flavonoids. This specific type of flavonoid, especially epigallocatechin gallate (EGCG), prevents the formation and growth of tumors.4 Normal cells take both complex and varying pathways to develop into malignant cells, but there are three crucial stages in the path to malignancy. In the initiation stage, undesirable mutations in the chromosome form due to exposure to carcinogenic substances or radiation. In the second stage of promotion, the mutation is translated and transcribed to the cytoplasm and cell membrane. The last stage is progression, during which cancer cells proliferate. By this point, accumulated mutations in chromosomes produce many genetic alterations that promote uncontrollable growth. While the numerous stages of cancer progression may complicate the search of one specific cure, they provide equal number of opportunities for regulation of carcinogenesis.5

The polyphenol substituents found in tea can suppress cancer at various stages in its progression. First, tea can prevent initiation by inactivating or eliminating the mutagens that can potentially damage the cell DNA. Potential mutagens are surprisingly common in our environment.5 Every day, we are exposed to processes that introduce dangerous reactive oxygen species (ROS) such as hydrogen peroxide and oxygen radicals that can react with DNA and induce detrimental mutations.1 Common ionizing radiation (UV and X-rays) as well as tobacco are well-documented mutagens as well. The flavonoids contained in tea are natural scavengers that destroy these free oxygen radicals.1 Catechin, a type of flavonoid, is especially effective at reducing free radicals by binding to ROS as well as to ferric ions, which are required to create ROS.6 Polyphenols of green tea can also competitively inhibit intermediates of heterocyclic aromatic amines, a new class of carcinogens, thus reducing the danger of accumulating DNA-damaging material.1 Finally, the chemical structure of the polyphenols in tea has strong affinity toward carcinogens, enabling them to bind to and neutralize the harmful substances.6 By blocking common cancer-initiating factors, tea lowers the chance of genetic mutations that may result in a tumor.

Substances in green tea can also prevent cancer by blocking angiogenesis, essentially starving the tumor cells.1 Angiogenesis is the formation of network of blood vessels through cancerous growths. In smaller tumors, cancer cells can use simple diffusion to transport necessary oxygen and nutrients. However, as the number of accumulated cells increase, tumor cells send signals to surrounding host tissues to produce the proteins necessary for blood vessel generation. These blood vessels supply large amounts of oxygen and nutrients that are unavailable through passive diffusion. Catechins in green tea stop angiogenesis by interfering with the tumor cell signals. EGCG has been shown to inhibit epidermal growth factor receptor, and thus production of vascular endothelial growth factor (VEGF), which is in charge of initiating angiogenic blood vessel formation.1 Further studies have shown direct inhibition of VEGF transcription and VEGF promoter activity in breast cancer cells by green tea extract (GTE) and EGCG.4-6 GTE also suppresses production of protein kinase C, which regulates VEGF as well. By inhibiting the signal pathway to blood vessel formation, green tea is able to reduce the progression of angiogenesis.

Another role of tea includes preventing metastasis, which is the most common cause of cancer-related mortality.1 Metastasis represents the full development of a tumor, in which the boundary that enclosed the cancer is broken and the tumor freely migrates to other parts of the body. Green tea’s flavonoids prevent degradation of membranes and proteins on the cell surface that promotes anchorage.1 Once base membranes and proteins that anchor cells to specific locations disappear, tumor cells are unfettered. EGCG in green tea has been shown to block metastasis by inhibition of membrane type 1 matrix metalloproteinase (MMP), which in turn restrains MMP-2, an enzyme crucial to degradation of the extracellular matrix. In experiments, a mixture of EGCG and ascorbic acid showed a significant suppression of metastasis by 65.9%.1

Finally, tea can prevent the unregulated proliferation of cancer cells that drives tumor formation and metastasis. Apoptosis, or the self-destruction of a cell, is actually a common and natural biological process. When a cell loses the ability to undergo apoptosis, it becomes potentially cancerous. Increasing apoptosis in cancer cells should restore balance and eliminate unrequired and harmful cells in the body. The problem lies in specifically inducing apoptosis of cancer cells without harming the normal cells, but research has shown tea’s potential in the selective promotion of apoptosis. In an experiment involving human papillomavirus 16-associated cervical cancer cells, EGCG inhibited cell growth by promoting apoptosis and cell cycle arrest.1 In head and neck carcinoma cells, EGCG also increased the percentage of cells at phase G1, the initial growth cycle of the cell, and induced apoptosis.1 Similar results were found by adding the extracted water-soluble fraction from green tea to mouse epidermal cells JB6, which both inhibited carcinogenesis and induced apoptosis.5

The extensive evidence presented here illustrates the cancer-preventive and inhibitory effects of green tea. However, we must consider that most of the data were collected through in vitro and in vivo experiments. Clinical trials with human beings have yet to confirm the preventive effects of tea polyphenol against cancer.5 Current research does not present significant evidence to determine the true effects of tea. On the other hand, a negative correlation has been observed between green tea consumption and cancer mortality along with general mortality rate in Japanese populations.5 In general, increasing the amount of green tea consumed per day indicated a reduced chance of cancer. These results suggest that tea, even with its vast number of health benefits, is not a cureall. In conjunction with regular exercise and vegetables with each meal, however, many diseases can be prevented. By drinking tea, one can partake in a tradition passed down for centuries while keeping the body healthy.


  1. Jain, N. K. et al. Protective Effects of Tea on Human Health; CAB International: Cambridge, 2006.
  2. Borgwardt, S. et al. Eur. J. Clin. Nutr. 2012, 66, 1187-1192.
  3. Zhang, Q. et al. Nutr. J. 2013, 12, 84.
  4. Dulloo, A. G. et al. Am. J. Clin. Nutr. 1999, 70, 1040-1045.
  5. Kuroda, Y. et al. Health Effects of Tea and Its Catechins; Kluwer Academic/Plenum Publishers: New York, 2004.
  6. Yammamoto, T. et al. Chemistry and Applications of Green Tea; CRC Press LLC: New York, 1997.