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Questions and Answers about CRISPR

Q: What is “CRISPR”?

A: “CRISPR” (pronounced “crisper”) stands for Clustered Regularly Interspaced Short Palindromic Repeats, which are the hallmark of a bacterial defense system which forms the basis for the popular CRISPR-Cas9 genome editing technology. In the field of genome engineering, the term “CRISPR” is often used loosely to refer to the entire CRISPR-Cas9 system, which can be programmed to target specific stretches of genetic code and to edit DNA at precise locations. These tools allow researchers to permanently modify genes in living cells and organisms and, in the future, may make it possible to correct mutations at precise locations in the human genome to treat genetic causes of disease. In September 2015, the Zhang lab demonstrated successful harnessing of a different CRISPR system for genome editing, called CRISPR-Cpf1, which has the potential for even simpler and more precise genome engineering.

Q: Where do CRISPRs come from?

A: CRISPRs were first discovered in archaea (and later in bacteria), by Francisco Mojica, a scientists at the University of Alicante in Spain. He proposed that CRISPRs serve as part of the bacterial immune system, defending against invading viruses. They consist of repeating sequences of genetic code, interrupted by “spacer” sequences – remnants of genetic code from past invaders. The system serves as a genetic memory that helps the cell detect and destroy invaders (called “bacteriophage”) when they return. Mojica’s theory was experimentally demonstrated in 2007 by a team of scientists led by Philippe Horvath.

In January 2013, Feng Zhang at the Broad Institute and MIT published the first method to engineer CRISPR to edit the genome in mouse and human cells.

Q: How does the system work?

A: CRISPR “spacer” sequences are transcribed into short RNA sequences (“CRISPR RNAs” or “crRNA”) capable of guiding the system to matching sequences of DNA. When the target DNA is found, Cas9 – one of the enzymes produced by the CRISPR system – binds to the DNA and cuts it, shutting the targeted gene off. Using modified versions of Cas9, researchers can activate gene expression instead of cutting the DNA. These techniques allow researchers to study the gene’s function.

Research also suggests that CRISPR-Cas9 can be used to target and modify “typos” in the three-billion-letter sequence of the human genome in an effort to treat genetic disease.

An artist's depiction of the CRISPR system in action.
Illustration by Stephen Dixon

Q: How does CRISPR-Cas9 compare to other genome editing tools?

A: CRISPR-Cas9 is proving to be an efficient and customizable alternative to other existing genome editing tools. Since the CRISPR-Cas9 system itself is capable of cutting DNA strands, CRISPRs do not need to be paired with separate cleaving enzymes as other tools do. They can also easily be matched with tailor-made “guide” RNA (gRNA) sequences designed to lead them to their DNA targets. Tens of thousands of such gRNA sequences have already been created and are available to the research community. CRISPR-Cas9 can also be used to target multiple genes simultaneously, which is another advantage that sets it apart from other gene-editing tools.

CRISPR-Cpf1 differs in several important ways from the previously described Cas9, with significant implications for research and therapeutics.

First: In its natural form, the DNA-cutting enzyme Cas9 forms a complex with two small RNAs, both of which are required for the cutting activity. The Cpf1 system is simpler in that it requires only a single RNA. The Cpf1 enzyme is also smaller than the standard SpCas9, making it easier to deliver into cells and tissues.

Second, and perhaps most significantly: Cpf1 cuts DNA in a different manner than Cas9. When the Cas9 complex cuts DNA, it cuts both strands at the same place, leaving ‘blunt ends’ that often undergo mutations as they are rejoined. With the Cpf1 complex the cuts in the two strands are offset, leaving short overhangs on the exposed ends. This is expected to help with precise insertion, allowing researchers to integrate a piece of DNA more efficiently and accurately.

Third: Cpf1 cuts far away from the recognition site, meaning that even if the targeted gene becomes mutated at the cut site, it can likely still be re-cut, allowing multiple opportunities for correct editing to occur.

Fourth: the Cpf1 system provides new flexibility in choosing target sites. Like Cas9, the Cpf1 complex must first attach to a short sequence known as a PAM, and targets must be chosen that are adjacent to naturally occurring PAM sequences. The Cpf1 complex recognizes very different PAM sequences from those of Cas9. This could be an advantage in targeting some genomes, such as in the malaria parasite as well as in humans.