CRISPR Background CRISPR Update
Posted: May 27, 2018 at 10:43 pm
Targeted gene editing began with the discovery of zinc finger proteins in the 1980s and continued to improve through the 1990s and early 2000s with the discovery of Transcription activator-like effector nucleases, or TALENS (1, 2). Both of these techniques rely on complex protein structures being engineered to target specific DNA sequences and containing a fused nuclease that nicks a single strand of the DNA duplex. In order to induce the double stranded break (DSB) needed for non-homologous end joining (NHEJ) or homology directed repair (HDR) two zinc finger or TALEN proteins are needed, each targeting one strand of the DNA duplex. While these techniques are reliable, the challenges in designing the protein structures needed to target specific DNA sequences limited their widespread adoption. In 2012 the CRISPR/Cas system was found to target and cut specific DNA sequences using only a nuclease and RNAs to target specific DNA sequences (3). The ease with which this system allows for targeting any gene has set off a new era in targeted gene editing.
Clustered Regular Interspaced Palindromic Repeats, or CRISPRs, were originally identified in the late 1980s in bacteria as short segments of repeating DNA separated by unique spacer sequences however their significance was originally, not known (4). It was not until the early 2000s that the term CRISPR was coined and specific genes, named CRISPR-Associated genes, or Cas genes, were identified (5). Throughout the next decade it was found that the unique spacer sequences were homologous to phage DNA and that certain Cas proteins (i.e. Cas9) used transcribed CRISPR RNA to target and cleave phage DNA, thus acting as an adaptive immune system for bacteria (3, 610). The CRISPR system is composed of two RNA components, crRNA and tracrRNA. Both are transcribed and are required for Cas9 cleavage activity (7). The crRNA is the RNA moiety that targets a specific gene sequence; it contains the transcribed unique spacer RNA as well as a palindromic repeat. The tracrRNA contains a palindromic repeat (the complementary sequence to the crRNA) and a region that can bind to Cas9. Upon duplexing of the crRNA and tracrRNA, this RNA complex can join with Cas9 to target DNA complementary to the unique spacer region of the crRNA (3). Once the crRNA forms a duplex with DNA and the PAM sequence is engaged, Cas9 will cut both strands of the DNA resulting in a double stranded break (DSB), thereby inducing the host DNA repair mechanisms.
After cleavage, DNA can by repaired one of two ways. The simplest, most efficient repair mechanism is referred to as Non-Homologous End-Joining (NHEJ) repair and is the result of enzymes adding and/or removing DNA bases at random to repair the break. This process can result in mutations, by either introducing a premature stop codon or by causing a frameshift mutation. Either one of these mutations ultimately results in a non-functional gene product. NHEJ is routinely used when researchers want to knockout a specific gene. Less efficient than NHEJ is Homology Directed Repair (HDR). HDR is used to insert/knockout genes or to make a specific change at a DSB. In addition to needing the CRISPR/Cas9 machinery, HDR requires a sequence of DNA whose ends are homologous to the ends of the DSB. After inducing a DSB, the cell inserts the new sequence through homologous recombination. To induce specific mutations in cells lines, addition of a donor DNA is needed.
In 2012 Jennifer Doundas group at University of California-Berkley characterized the activity of Cas9 and found that the two RNA component of Cas9 could be modified into a single strand of RNA. This new RNA fragment was coined the guide RNA (gRNA), also known as a single guide RNA. The gRNA is composed of a truncated tracrRNA sequence coined the scaffold sequence fused to a ~20 nucleotide user defined spacer or targeting sequence (3). This system can theoretically be used to target any sequence in a genome provided it meets two conditions. First, the sequence must be unique when compared to the rest of the genome and second, the target sequence has to be immediately followed by the Protospacer Adjacent Motif (PAM). The PAM is a 3-5 nucleotide sequence that is required for Cas cleavage activity. Cas9 has a three nucleotide PAM NGG while other Cas proteins have been identified with different PAM sequences (11). Additionally, protein engineering has been used to create Cas9 variants with different PAM sequences thus expanding the number of genomic targets possible.
Identification of CRISPR mutations depends on which repair mechanism is employed. When large genes are inserted by HDR, PCR amplification of the transgene can easily identify which lines are positive for the desired event. When HDR is used to repair small sections of DNA that do not result in large insertions, sequencing or heteroduplex cleavage are used to identify the changes. A DSB repaired via NHEJ can be detected using a heteroduplexing and endonuclease assay such as T7EI or Surveyor. Upon heteroduplexing of the mutated sequence with a wild-typesequence, T7EI or Surveyor can cleave at the mismatched DNA bases. Successfully modified sequences are then identified by comparing the fragment sizes produced by the assay with the theoretical fragment size of the CRISPR targeted sequence. The ease at which CRISPR/Cas systems can be programed to target virtually any gene in any genome potentially allows for widespread adoption in a number of industries and applications. . Right now, CRISPR is being used to understand how different genes impact human disease through the use of several model animal systems. It is also being used to engineer the next generation of production crops and animals. In the more immediate future CRISPR gene editing may be used to potentially fight widespread zoonotic diseases such as malaria. The applications are endless. While no one can be certain how far reaching the impact CRISPR technology will be, it has undoubtedly revolutionized molecular biology.
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CRISPR Background CRISPR Update
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