A Brief History of CRISPR

DNA strand with explosive break

Maybe you heard the Fresh Air interview with Michael Specter about gene editing:

Or you’re studying genetics in school and you’re biocurious. Interested in the field of synthetic biology? Thinking about editing your first genome? You’ve undoubtedly heard of CRISPR, the advanced, current generation genome editing technology. Currently, it is mentioned in over 4800 articles in PubMed, with exponential adoption rates over the last three years.

If you are just getting started, or even know a thing or two about the technology, Twist Bioscience offers a science history lesson on the development of CRISPR based genetic engineering via instrumental scientific publications.

Prokaryotic genomes contain well-organized repeats
Jansen et al., 2002

Scientists had previously noted that many bacterial and archaeal genomes contained distinct repeated motifs of <50bp that were clearly, neatly, and consistently ordered.

Order implies function, but their function was baffling — they were non-coding for one, and the pattern kept showing up in different species, each with its own unique, repetitive sequence that was often highly diverged from other species with the same pattern.

Efforts to find a solution to their function came in the paper above, kickstarting the field by naming the repeat regions “Clustered Regularly Interspaced Short Palindromic Repeats” (CRISPR) and documenting the existence a number of CRISPR-associated genes (named the Cas family) adjacent to these repeats. The CRISPR-Cas paradigm was born.

Sequences in between these repeats are surprisingly foreign
Mojica et al., 2005

CRISPR were always found interspersed with what seemed like totally arbitrary sequence, also <50bp in length. A CRISPR locus looks like this:


Mojica et al. sequenced 4500 CRISPR sequences from 67 strains representing both bacteria and archaea, comparing these sequences against repositories within GenBank.

What they found was astounding. The sequences matched a mixture of bacteriophage (viruses that infect bacteria) sequences, invasive plasmid (weapons used by bacteria to destroy other bacteria) sequences, and personal genomic sequences which had been sequestered into CRISPR interspacing regions.

Eukaryotes are not the only organisms to have an adaptive immune system
Barrangou et al., 2007

The authors of the previous paper noticed that Sulfolobus solfataricus, anarchaea that grows in hot springs, was naturally immune to a virus called SIRV. It also had SIRV DNA in its CRISPR spacers, noteworthy in that viruses use DNA as their weapon to infect hosts. It was hypothesized that CRISPR was a form of bacterial adaptive immunity against viral attack.

Barrangou et al., showed that subjecting bacteria to viral attack until it became resistant led to the attacking virus’ DNA being introduced into the CRISPR interspacing regions. To account for false positives, they removed the spacers containing viral DNA from the resistant strain, and subjected it to viral attack once again. Resistance was instantly lost.

Cas uses CRISPR to become a guided missile
Garneau et al., 2010 and Deltcheva et al., 2011

Given that CRISPR and spacer DNA provides immunity to viruses, scientists began digging for the precise mechanism that leads to one genetic element destroying another. Together both featured papers show that CRISPR/Cas uses the information in CRISPR spacers as coordinates for cutting up invading DNA sequences.

Viral DNA challenged with CRISPR/Cas of a resistant strain was always cleaved within the sequence that matched the spacer. This cleavage always occurred at a specific distance from a recognized CRISPR sequence motif that was always consistent between spacers of any particular species (the Protospacer Adjacent Motif, or PAM).

Virus resistant-bacteria produce lots of RNA from two distinct regions in the CRISPR/Cas system. One is the CRISPR spacer itself (crRNA), however the other is just outside of the CRISPR repeats, near where the Cas genes are found (tracrRNA). Both RNA fragments together were necessary to cleave viral DNA alongside the endonuclease protein Cas9.

If Cas9 is CRISPR RNA guided, and we can engineer DNA sequences
Jinek et al., 2012

This featured study could be a good candidate for the greatest biological advance in the last five years.

It was a one-two punch knockout, with an incredible leap that revolutionized not just synthetic biology, but genetic engineering, personalized medicine, agricultural science, genetics, and cell biology, to name just a few.

Jinek et al. demonstrated that because crRNA and tracrRNA are complimentary sequences, they bind into a double strand. This double strand guides the Cas9 to the complimentary strand in the invasive DNA starting at the PAM. The Cas9, which contains two different DNA cutting domains, unwinds the DNA into two strands, and then creates a blunt break in the invading DNA.

The authors (Jinek, Chylinski, Fonfara and Charpentier) showed that linking the crRNA and the tracrRNA into a new molecule they named guide RNA (gRNA), they could simplify the system. Any gRNA sequence could be synthesized to facilitate Cas9-mediated blunt ended cleavage of any DNA sequence. This gives researchers precise control to cut anywhere in an organism’s genome, allowing researchers to engineer introduce or knock out genes of within any selected organism’s genome with relative ease.

Gene Engineering is not only made possible with CRISPR; it’s six times faster!

Inui et al., 2014

Previous protocols to develop mice for genetic testing required about six months of mutagenesis and cross breeding. With CRISPR/Cas9, this process can be shortened to four weeks!

Wait, could CRISPR target the wrong sequence?

Fu et al., 2013 and Hsu et al., 2013

Even when gRNA mismatches a few bases, Cas9 can still cut DNA. The impact of mismatched cleavage had to be fully assessed and quantified to mitigate any possible negative impact to consider future genome engineering processes.

These papers identify the infrequency with which Cas9 does fire stray bullets and importantly these papers bring the ethics debate to the forefront of the field, opening the doors for extensive discussion among those actively working in the space.

Making Cas9 worse makes Cas9 better

Kleinstiver et al., 2016

As an answer to the concerns regarding gRNA mismatch, many studies attempted to make Cas9 more specific for its target, reducing its off-target effects. The brand new research in this featured article offers an attractive solution that could be easily utilized in any CRISPR project.

Cas9 has four different domains that interact with DNA. The authors posit that this increases the binding energy of Cas9. This excess energy allows it to bind to the target sequence even if there are up to five mismatches. Kleinstiver et al. took this idea, and weakened these DNA interacting domains. This reduces the binding energy, meaning Cas9 does not cling on to the target sequence as tightly.

The effect? In order to have a high enough binding energy to produce a cut, the gRNA and the target DNA must be an exact match. High-throughput sequencing showed that this high fidelity Cas9 produced zero detectable off-target effects.

Gene drives could wipe out deadly diseases

Gantz and Bier, 2015

CRISPR/Cas9-mediated “gene drives” could allow almost every offspring of a gene driven organism to display a desired phenotype, as opposed to only half of the offspring in a non-driven organism. This occurs because each offspring inherits their own gene drive to integrate the necessary modifications. It is the gene drive that is passed onto every offspring.

There is hope that this technology could be used in wild mosquito populations to eliminate malaria, dengue fever and the Zika virus, which are responsible for thousands of deaths worldwide annually.

CRISPR/Cas9 dipped its toes into human genome editing

Liang et al., 2015 and Bosley et al., 2015

This is a paper that scientists simultaneously expected and dreaded. It is the first published example of CRISPR/Cas9 used on human germline cells. While these cells were totally non-viable to begin with, their modification led to an explosion of debate, culminating in an international summit, with over 400 international experts. The result was a refreshed mindfulness around the ethics surrounding human genome editing, as well as a respect for and excitement over the positive impact possible through this powerful technology — with a stated objective for additional multi-faceted dialogue.

‘Dead’ Cas9 makes the CRISPR/Cas9 system even more alive

Qi et al., 2013, Bickard et al. 2013 and Dominguez et al., 2015

Cas9 that is precisely targeted to a genomic region of interest can potentially offer more applications than just DNA cleavage. These two papers show that by de-activating the cleavage domains in Cas9 to make ‘dead’ Cas9 (dCas9) and fusing dCas9 with other DNA-acting proteins, the DNA-acting proteins can be precisely guided to specific genomic regions. Potential targets are proteins that facilitate chromatin re-modelling, methylation or expression activators and repressors. This could set the stage for using a patient’s own stem cells to repair physical or autoimmune damage without rejection.

CRISPR fusion proteins can accurately modify single amino acids without cutting

Komor et al., 2016

Dead Cas9 has been fused to protein called a cytidine deaminase, allowing controlled editing of single bases. Cytidine deaminase is able to turn a cytosine © nucleic acid to a Thymine (T). With this fusion protein, potentially any cytosine in the genome can be converted to a thymine. This is the first step in being able to precisely control genome modification, which ultimately has potential to correct point mutations that are the cause of many genetic diseases, as well as some cancers.

The status quo

CRISPR has exploded in the last four years, seeing exponential growth. Towards the end of 2015 there were five papers a day being published in the field. As of the date of this post in mid-January 2017, over 250 new papers have been submitted to the PubMed journal database. We are excited to see where this amazing technology will take us in the coming year.

This post originally appeared as a series in the Twist Bioscience blog DNA by Design.