Origins of CRISPR: How it Came to Be

Gene editing isn’t a new concept and has been around for decades. In its infancy, gene editing was time-consuming, inaccurate and costly. That all changed when a revolutionary technique called CRISPR took the science community by storm and has since made the process more affordable, efficient and accurate. 

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) sequences were first discovered in the E. coli genome in 1987, but it wasn’t until 2007 that their role as a bacteriophage defense was decoded. 

More technically, Cas9 is a dual RNA-guided DNA endonuclease enzyme associated with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) adaptive immune system in Streptococcus pyogenes.[3][4] S. pyogenes utilizes CRISPR to memorize and Cas9 to later interrogate and cleave foreign DNA, such as invading bacteriophage DNA or plasmid DNA.[4][5][6][7] Cas9 performs this interrogation by unwinding foreign DNA and checking for sites complementary to the 20 basepair spacer region of the guide RNA. If the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA. In this sense, the CRISPR-Cas9 mechanism has a number of parallels with the RNA interference (RNAi) mechanism in eukaryotes.

CRISPR/Cas9 is a gene-editing technology that involves two essential components: a guide RNA to match the desired target gene, and Cas9 (CRISPR-associated protein 9)—an endonuclease that causes a double-stranded DNA break, allowing modifications to the genome

CRISPR gene editing has already changed the way scientists do research. But the technology could also hold great power used as a treatment for human diseases. In theory, CRISPR technology could let us edit any genetic mutation at will, curing any disease with a genetic origin. In practice, we are just at the beginning of the development of CRISPR as a therapy and there are still many unknowns.

CRISPR History and Development

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) sequences were initially discovered in the E. coli genome in 1987, but their function as a safeguard against bacteriophages was not elucidated until 2007. Scientists hypothesized that prokaryotes used CRISPR as part of an adaptive immune system – utilizing various CRISPR-associated (Cas) genes to not only store a record of invading phages but also to destroy the phages upon re-exposure.

More specifically, specialized Cas proteins snip foreign DNA into small fragments approximately 20 bp in length and paste them into contiguous stretches of DNA known as CRISPR arrays. Separate Cas proteins then express and process the CRISPR loci to generate CRISPR RNAs (crRNAs). Through sequence homology, these crRNAs guide a Cas nuclease to the specified exogenous genetic material, which must also contain a species-specific sequence known as a protospacer adjacent motif (PAM). The CRISPR complex binds to the foreign DNA and cleaves it to destroy the invader. 

The discovery of the CRISPR-Cas microbial adaptive immune system and its ongoing development into a genome editing tool represents the work of many scientists from around the world. This timeline presents a concise history of the seminal contributions and the scientists who pushed this field forward, from the initial discovery to the first demonstrations of CRISPR-mediated genome editing.  For a narrative perspective of the history of CRISPR research, read “The Heroes of CRISPR,” by Eric S. Lander, in the January 14, 2016 edition of Cell.

CRISPR-Cas9 was first used as a gene-editing tool in 2012. In just a few years, the technology has exploded in popularity thanks to its promise of making gene editing much faster, cheaper, and easier than ever before.

This prokaryotic immune system is especially amenable to genome engineering, offering flexibility and easy multiplexing/scaling. Prokaryotes have long utilized CRISPR as a powerful defensive strategy against viral invaders, and this system is proving to be just as useful (if not more so) for research applications, eclipsing past genome engineering technologies like zinc finger nucleases (ZFNs) and TALENs.

Discovery of CRISPR and its function

1993 – 2005 — Francisco Mojica, University of Alicante, Spain

Francisco Mojica was the first researcher to characterize what is now called a CRISPR locus, reported in 1993. He worked on them throughout the 1990s, and in 2000, he recognized that what had been reported as disparate repeat sequences actually shared a common set of features, now known to be hallmarks of CRISPR sequences (he coined the term CRISPR through correspondence with Ruud Jansen, who first used the term in print in 2002). In 2005 he reported that these sequences matched snippets from the genomes of bacteriophage (Mojica et al., 2005). This finding led him to hypothesize, correctly, that CRISPR is an adaptive immune system. Another group, working independently, published similar findings around this same time (Pourcel et al., 2005)

Discovery of Cas9 and PAM

May, 2005 — Alexander Bolotin, French National Institute for Agricultural Research (INRA)

Bolotin was studying the bacteria Streptococcus thermophilus, which had just been sequenced, revealing an unusual CRISPR locus (Bolotin et al., 2005). Although the CRISPR array was similar to previously reported systems, it lacked some of the known cas genes and instead contained novel cas genes, including one encoding a large protein they predicted to have nuclease activity, which is now known as Cas9. Furthermore, they noted that the spacers, which have homology to viral genes, all share a common sequence at one end. This sequence, the protospacer adjacent motif (PAM), is required for target recognition.

Diseases CRISPR Technology Could Cure

  1. Remove malaria from mosquitos. Scientists have created mosquitoes that are resistant to malaria by deleting a segment of mosquito DNA. The altered mosquitoes pass on the resistance genes to 99 percent of their offspring, even when they mated with normal mosquitos.
  1. Treating Alzheimer’s disease. CRISPR-based platforms have been developed to identify the genes controlling the cellular processes that lead to neurodegenerative diseases like Alzheimer’s and Parkinson’s, hopefully leading to new treatments.
  1. Treating HIV. The HIV virus inserts its DNA into the cells of the human host. CRISPR has been successful in removing the virus’s DNA from the patient’s genome. Other genetic sequences will likely be found that eliminate HIV, herpes, hepatitis, and other dangerous viruses.
  1. Develop new drugs. Pharmaceutical companies such as Bayer AG are investing hundreds of millions of dollars to develop CRISPR-based drugs to treat heart disease, blood disorders, and blindness.
  1. Livestock. CRISPR/Cas9 has been utilized in China to delete genes in livestock that inhibit muscle and hair growth to grow larger stock for the country’s commercial meat and wool industries. This could become a common way in the future to expand livestock industries.
  1. Agricultural crops. Researchers are using CRISPR to discover new ways to improve crop disease resistance and environmental stress tolerance in plants. If successful, this could result in new crops to help feed the global population.
  1. Develop new cancer treatments. CRISPR can modify immune cells to make them more effective at targeting and destroying cancer cells. CRISPR can also be used to evaluate how genes can be studied to determine their sensitivity to new anti-cancer drugs, thereby developing a personalized treatment plan with the best possibility of success.
  1. Reduce our need for plastic. CRISPR can be used to manipulate a type of yeast that transforms sugars into hydrocarbons, which can be used to make plastic—greatly reducing the need to rely on petroleum-based resources for plastics, easing stress on the environment.

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illustration of insertion on a molecule