Double Strand DNA Breaks Introduced by CRISPR-Cas9 Allows Further Genetic Manipulation By Exploiting Endogenous DNA Repair Mechanisms.

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The discovery of clustered DNA repeats occurred independently in three parts of the world. The first description of what would later be called CRISPR is from Osaka University researcher Yoshizumi Ishino and his colleagues in 1987. They accidentally cloned part of a CRISPR together with the iap gene, the target of interest. The organization of the repeats was unusual because repeated sequences are typically arranged consecutively along DNA. They studied the relation of “iap“ to the bacterium E. coli. The function of the interrupted clustered repeats was not known at the time. In 1993 researchers of Mycobacterium tuberculosis in the Netherlands published two articles about a cluster of interrupted direct repeats (DR) in this bacterium. These researchers recognized the diversity of the DR-intervening sequences among different strains of M. tuberculosis and used this property to design a typing method that was named spoligotyping, which is still in use today. At the same time, repeats were observed in the archaeal organisms of Haloferax and Haloarcula species, and their function was studied by Francisco Mojica at the University of Alicante in Spain. Although his hypothesis turned out to be wrong, Mojica’s supervisor surmised at the time that the clustered repeats had a role in correctly segregating replicated DNA into daughter cells during cell division because plasmids and chromosomes with identical repeat arrays could not coexist in Haloferax volcanii. Transcription of the interrupted repeats was also noted for the first time. By 2000, Mojica performed a survey of scientific literature and one of his students a search in published genomes with a program devised by himself. They found interrupted repeats in 20 species of microbes, and it was the first time different repeats with the same properties were identified as belonging to the same family, not yet known as CRISPR. In 2001, Mojica and Ruud Jansen, who was searching for additional interrupted repeats, proposed the acronym CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to alleviate the confusion stemming from the numerous acronyms used to describe the sequences in the scientific literature.


CRISPR-Associated Systems


A major addition to the understanding of CRISPR came with Jansen’s observation that the prokaryote repeat cluster was accompanied by a set of homologous genes that make up CRISPR-associated systems or cas genes. Four cas genes (cas 1 – 4) were initially recognized. The Cas proteins showed helicase and nuclease motifs, suggesting a role in the dynamic structure of the CRISPR loci. In this publication the acronym CRISPR was coined as the universal name of this pattern. However, the CRISPR function remained enigmatic.


Simplified diagram of a CRISPR locus. The three major components of a CRISPR locus are shown: cas genes, a leader sequence, and a repeat-spacer array. Repeats are shown as gray boxes and spacers are colored bars. The arrangement of the three components is not always as shown. In addition, several CRISPRs with similar sequences can be present in a single genome, only one of which is associated with cas genes. In 2005, three independent research groups showed that some CRISPR spacers are derived from phage DNA and extrachromosomal DNA such as plasmids. In effect, the spacers are fragments of DNA gathered from viruses that previously tried to attack the cell. The source of the spacers was a sign that the CRISPR/cas system could have a role in adaptive immunity in bacteria. All three studies proposing this idea were initially rejected by high-profile journals, but eventually appeared in other journals. The first publication proposing a role of CRISPR-Cas in microbial immunity, by the researchers at the University of Alicante, predicted a role for the RNA transcript of spacers on target recognition in a mechanism that could be analogous to the RNA interference system used by eukaryotic cells. This hypothesis had already been defended in a pre-doc examination and one scientific meeting in 2004. Koonin and colleagues extended this RNA interference hypothesis by proposing mechanisms of action for the different CRISPR-Cas subtypes according to the predicted function of their proteins.


Experimental work by several groups revealed the basic mechanisms of CRISPR-Cas immunity. In 2007 the first experimental evidence that CRISPR was an adaptive immune system was published. A CRISPR region in Streptococcus thermophilus acquired spacers from the DNA of an infecting bacteriophage. The researchers manipulated the resistance of S. thermophilus to phage by adding and deleting spacers whose sequence matched those found in the tested phages. In 2008, Brouns and Van der Oost identified a complex of Cas proteins (called Cascade) that in E. coli cut the CRISPR RNA precursor within the repeats into mature spacer-containing RNA molecules (crRNA), which remained bound to the protein complex. Moreover, it was found that Cascade, crRNA and an helicase/nuclease (Cas3) were required to provide a bacterial host with immunity against infection by a DNA virus. By designing an anti-virus CRISPR, they demonstrated that two orientations of the crRNA (sense/antisense) provided immunity, indicating that the crRNA guides were targeting dsDNA. That year Marraffini and Sontheimer indeed confirmed that a CRISPR sequence of S. epidermidis targeted DNA and not RNA to prevent conjugation. This finding was at odds with the proposed RNA-interference-like mechanism of CRISPR-Cas immunity, although a CRISPR-Cas system that targets foreign RNA was later found in Pyrococcus furiosus. A 2010 study showed that CRISPR-Cas cuts both strands of phage and plasmid DNA in S. thermophilus.




Researchers studied a simpler CRISPR system from Streptococcus pyogenes that relies on the protein Cas9. The Cas9 endonuclease is a four-component system that includes two small RNA molecules named CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). Jennifer Doudna and Emmanuelle Charpentier re-engineered the Cas9 endonuclease into a more manageable two-component system by fusing the two RNA molecules into a “single-guide RNA“ that, when combined with Cas9, could find and cut the DNA target specified by the guide RNA. By manipulating the nucleotide sequence of the guide RNA, the artificial Cas9 system could be programmed to target any DNA sequence for cleavage. Another group of collaborators comprising Siksnys together with Gasiunas, Barrangou and Horvath showed that Cas9 from the S. thermophilus CRISPR system can also be reprogrammed to target a site of their choosing by changing the sequence of its crRNA. These advances fueled efforts to edit genomes with the modified CRISPR-Cas9 system. Feng Zhang’s and George Church’s groups simultaneously described genome editing in human cell cultures using CRISPR-Cas9 for the first time. It has since been used in a wide range of organisms, including baker’s yeast (Saccharomyces cerevisiae), the opportunistic pathogen C. albicans, zebrafish (D. rerio), fruit flies (Drosophila melanogaster), nematodes (C. elegans), plants, mice, monkeys and human embryos.


CRISPR has been modified to make programmable transcription factors that allow scientists to target and activate or silence specific genes. The CRIPSR/Cas9 system has shown to make effective gene edits in Human tripronuclear zygotes first described in a 2015 paper by Chinese scientists P. Liang and Y. Xu. The system made a successful cleavage of mutant Beta-Hemoglobin (HBB) in 28 out of 54 embryos. 4 out of the 28 embryos were successfully recombined using a donor template given by the scientists. The scientists showed that during DNA recombination of the cleaved strand, the homologous endogenous sequence HBD competes with the exogenous donor template. DNA repair in human embryos is much more complicated and particular than in derived stem cells.




In 2015, the nuclease Cpf1 was discovered in the CRISPR/Cpf1 system of the bacterium Francisella novicida. Cpf1 showed several key differences from Cas9 including: causing a ?staggered’ cut in double stranded DNA as opposed to the ?blunt’ cut produced by Cas9, relying on a ?T rich’ PAM (providing alternate targeting sites to Cas9) and requiring only a CRISPR RNA (crRNA) for successful targeting. By contrast Cas9 requires both crRNA and a transactivating crRNA (tracrRNA). These differences may give Cpf1 some advantages over Cas9. For example, Cpf1’s small crRNAs are ideal for multiplexed genome editing, as more of them can be packaged in one vector than can Cas9’s sgRNAs. As well, the sticky 5′ overhangs left by Cpf1 can be used for DNA assembly that is much more target-specific than traditional Restriction Enzyme cloning. Finally, Cpf1 cleaves DNA 18-23 bp downstream from the PAM site. This means there is no disruption to the recognition sequence after repair, and so Cpf1 enables multiple rounds of DNA cleavage. By contrast, since Cas9 cuts only 3 bp upstream of the PAM site, the NHEJ pathway results in indel mutations which destroy the recognition sequence, thereby preventing further rounds of cutting. In theory, repeated rounds of DNA cleavage should cause an increased opportunity for the desired genomic editing to occur.



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