The History and Evolution of CRISPR

In the last few years, CRISPR has exploded into the biological space—and it seems everyone is pondering, performing, and publishing on CRISPR. A revolutionary gene-editing tool repurposed from a bacterial adaptive immune system, CRISPR is allowing researchers to make targeted, specific changes to the genome relatively easily and cheaply, opening up a raft of applications from basic gene function analysis to genome-wide functional genomic screening, as well as potentially moving into the clinic as a tool for gene therapy. This article tells the story of CRISPR—from its discovery from accidental cloning and characterization as a bacterial immune system, to repurposing as the powerful gene-editing tool we know today.

Discovery of the CRISPR Locus

The story of CRISPR begins in Japan in 1987 with the discovery of an unusual repetitive DNA sequence in E. coli, which was accidentally cloned along with the gene responsible for the isozyme conversion of alkaline phosphatase.¹ Rather than the usual motifs found in tandem repeats, these were regularly spaced sequences separated by five, non-repetitive sequences, later termed protospacers. The biological significance of these repeat elements was unknown, but they kept cropping up in the genomes of several different archaea and bacterial species. In 2000, with advancements in genomic sequencing and bioinformatic analysis, Mojica et al., found this clustered array was highly conserved across multiple evolutionary distinct bacterial genomes and identified several clusters of genes associated with the locus that were actively transcribed.² Taken together, this suggested a potentially significant biological function, and the locus was named Clustered Regularly Interspaced Short Palindromic Repeats, or CRISPR.³

It wasn’t until 2005 that the function of the CRISPR array was discovered, when three different labs independently reported that DNA within the spacer regions in the CRISPR array was homologous to extra chromosomal DNA found in bacteriophage and plasmids, and that they conferred resistance to infection.^4-6 From this, work soon led to the discovery that elements of the CRISPR array, consisting of protospacers, protospacer adjacent motifs (PAM), and CRISPR-associated genes (cas) with nuclease activity, work together to form a bacterial adaptive immune system, rather like RNAi, to protect prokaryotic cells from invading viruses and plasmids.⁷

CRISPR’s potential for gene editing

The CRISPR locus remained a subject of study for the next few years and three different types of CRISPR systems were identified and characterized, all with multiple subtypes—but distinguishable by their signature proteins: Cas3 for Type I, Cas9 for Type II, and Cas10 for Type III.

It was a collaboration between Emmanuelle Charpentier and Jennifer Doudna that then lead to the repurposing of the CRISPR-Cas system for gene editing. The simplicity of the Type II system—consisting of just a transactivating RNA (tracrRNA) together with a crRNA—made it the most tractable choice for development into a genome-editing tool. Charpentier and Doudna simplified the system further by fusing the crRNA and tracrRNA into a single guide RNA (sgRNA) and in 2012 showed the first evidence that Cas9 from Streptococcus pyogenes could be targeted to a specific locus to mediate double-strand DNA cleavage.⁸

The potential benefit as a molecular tool was not lost on the researchers and within six months of Charpentier and Doudna’s seminal work, this refined and repurposed CRISPR-Cas9 system was used in three separate studies to successfully edit bacterial and mammalian genomes ^{in vivo} and so a brand new, and incredibly powerful tool for editing genomes was created.^9-12

Applications of the CRISPR system

Previous gene-editing technologies, including Zinc finger nucleases (ZFNs) and TALENs, relied on expensive and complex protein engineering, but retargeting Cas9 requires only a change to the 20-nucleotide portion of the sgRNA complementary to the target loci (see takeaway box below). Consequently, gene editing became feasible for any researcher with access to basic molecular biology tools, and so the new CRISPR technology was rapidly adopted, with the number of publications featuring CRISPR-Cas9 quickly outnumbering those for ZFN and TALENs combined.

In addition to targeting single genes to create cell and animal knockout models, scientists adapted the technology to power genome-wide, functional genomic screens. By designing a guide (or multiple guides) against every gene in the genome and combining this with lentiviral-mediated genome integration and next-generation sequencing, scientists could determine which gene disruptions were driving sensitivity or resistance in a screening condition and do this without the problems of off-target or inefficient knockdown often observed with shRNA screening.^13-15

More recently scientists have begun to array multiple guides into single targeting vectors, enabling multiplexed gene editing—facilitating either rapid creation of more complex knockout models, or screening of dual guides during genomic screening.

Expanding the CRISPR toolkit

Initial work with CRISPR-Cas9 relied on the introduction of a double-strand break to facilitate gene knockouts but it wasn’t long until the CRISPR system was repurposed to allow for other modifications to gene expression.¹⁶ CRISPR interference, or CRISPRi, utilizes a catalytically inactive version of Cas9 (dCas9) targeted to promoter regions to inhibit gene expression, allowing targeted but reversible knockdown rather than knockout. Using a similar approach, dCas9 can also be used to activate gene expression with CRISPR activation, or CRISPRa. The ability to easily generate chimeric versions of dCas9 provides a flexible and modular system to recruit proteins to specific DNA target sites, allowing interrogation of regulatory regions, as well as approaches such as base editing and prime editing.

The future is CRISPR

The development of CRISPR-Cas9 as a gene-editing tool has revolutionized how scientists study gene function, leading to more robust data and a better understanding of biology—and subsequent development of the tool has expanded the scale of this revolution still further. The CRISPR system in its many forms remains easy to design and use, and therefore accessible to the wider scientific community—which promises many CRISPR-Cas9 facilitated discoveries are still to come.

How CRISPR-based gene editing works

The S. pyogenes CRISPR gene-editing system comprises of two components—the sgRNA and the Cas9 endonuclease. The Cas9 protein recognizes a 3’-NGG-5’ protospacer-associated motif or PAM site, which is ubiquitous throughout the genome, so there are plenty of sites that can be targeted. If the sequence of the sgRNA shares sufficient homology to the DNA sequence adjacent to the PAM site, the endonuclease activity of Cas9 is activated and a double-strand break (DSB) is introduced at the target loci.

The DSB then activates endogenous DNA repair pathways, such as Non-Homologous End Joining (NHEJ), which can be subject to error prone repair and so often results in a frameshift mutation and subsequent gene knockout at the target site. Alternatively, in the presence of an exogenous donor template, the Homology Direct Repair (HDR) pathway will be activated, allowing precise changes to the DNA sequence to be made.

References

1. Ishino, Y., et al. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J. Bacteriol. 169, 5429–5433 (1987)

2. Mojica, F. J. M., et al. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Molecular Microbiology 36, 244–246 (2000)

3. Jansen, R., et al. Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 43, 1565–1575 (2002)

4. Mojica, F. J. M., et al. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 60, 174–182 (2005)

5. Bolotin, A., et al. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151, 2551–2561 (2005).

6. Pourcel, C., et al. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151, 653–663 (2005)

7. Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science (80-). 315, 1709–1712 (2007)

8. Jinek, M. et al. A programmable dual-RNA-guided dna endonuclease in adaptive bacterial immunity. Science (80-. ) 337, 816–821 (2012)

9. Jiang, W., et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233–239 (2013)

10. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science (80-. ). 339, 819–823 (2013)

11. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–6 (2013)

12. Jinek, M. et al. RNA-programmed genome editing in human cells. Elife 2, e00471 (2013)

13. Wang, T. et al. Genetic screens in human cells using the CRISPR-Cas9 system. Science (New York, N.Y.) vol. 343,6166 (2014): 80-4. doi:10.1126/science.1246981

14. Zhou, Y. et al. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature vol. 509,7501 (2014): 487-91. doi:10.1038/nature13166

15. Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science (New York, N.Y.) vol. 343,6166 (2014): 84-87. doi:10.1126/science.1247005

16. Qi, Lei S et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell vol. 152,5 (2013): 1173-83. doi:10.1016/j.cell.2013.02.022