TechII-Genomics

CRISPR gene editing: A commercial timeline

In just three years, CRISPR gene editing has hurtled to the forefront of biological science – with vast potential in agribusiness, human health, and industrial biotechnology. And unlike many nascent biotechs, which take years to attract significant funding, CRISPR has already attracted a lot of commercial interest. 2015 was a banner year for the technology. One could wager that once this patent nonsense is behind us, CRISPR will accelerate forward at breakneck pace.

The Broad Institute has a comprehensive timeline of CRISPR’s scientific development. Scientific American just published a story that details CRISPR’s commercial timeline. Here it is, sequentially:

January 2015

  • Novartis signs two deals with Intellia Therapeutics and Caribou Biosciences to engineer immune cells and blood stem cells, meant for drug discovery research.
  • AstraZeneca signs four deals with the Wellcome Trust Sanger Institute, the Innovative Genomics Initiative, the Broad and Whitehead Institutes in Massachusetts, and Thermo Fisher Scientific. It’s meant for preclinical validation of new drug targets.

May 2015

  • Juno Therapeutics and Editas Medicine collaborate on next-gen CAR-T and TCR Cell therapies.

August 2015

October 2015

  • Vertex Pharmaceuticals and CRISPR Therapeutics sign a deal worth up to $2.6 billion.

December 2015

  • Bayer and CRISPR Therapeutics team up in a $335 million gene editing pact to develop new therapies for blood disorders, blindness and congenital heart disease.

January 2016

(Source: MedCityNews)

CRISPR Timeline

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.


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.

Hypothetical scheme of adaptive immunity

March, 2006 — Eugene Koonin, US National Center for Biotechnology Information, NIH

Koonin was studying clusters of orthologous groups of proteins by computational analysis and proposed a hypothetical scheme for CRISPR cascades as bacterial immune system based on inserts homologous to phage DNA in the natural spacer array, abandoning previous hypothesis that the Cas proteins might comprise a novel DNA repair system.

Experimental demonstration of adaptive immunity

March, 2007 — Philippe Horvath, Danisco France SAS

S. thermophilus is widely used in the dairy industry to make yogurt and cheese, and scientists at Danisco wanted to explore how it responds to phage attack, a common problem in industrial yogurt making. Horvath and colleagues showed experimentally that CRISPR systems are indeed an adaptive immune system: they integrate new phage DNA into the CRISPR array, which allows them to fight off the next wave of attacking phage (Barrangou et al., 2007). Furthermore, they showed that Cas9 is likely the only protein required for interference, the process by which the CRISPR system inactivates invading phage, details of which were not yet known.

Spacer sequences are transcribed into guide RNAs

August, 2008 — John van der Oost, University of Wageningen, Netherlands

Scientists soon began to fill in some of the details on exactly how CRISPR-Cas systems “interfere” with invading phage. The first piece of critical information came from John van der Oost and colleagues who showed that in E-scherichia coli, spacer sequences, which are derived from phage, are transcribed into small RNAs, termed CRISPR RNAs (crRNAs), that guide Cas proteins to the target DNA (Brouns et al., 2008).

CRISPR acts on DNA targets

December, 2008 — Luciano Marraffini and Erik Sontheimer, Northwestern University, Illinois

The next key piece in understanding the mechanism of interference came from Marraffini and Sontheimer, who elegantly demonstrated that the target molecule is DNA, not RNA (Marraffini and Sontheimer, 2008). This was somewhat surprising, as many people had considered CRISPR to be a parallel to eukaryotic RNAi silencing mechanisms, which target RNA. Marraffini and Sontheimer explicitly noted in their paper that this system could be a powerful tool if it could be transferred to non-bacterial systems. (It should be noted, however, that a different type of CRISPR system can target RNA (Hale et al., 2009)).

Cas9 cleaves target DNA

December, 2010 — Sylvain Moineau, University of Laval, Quebec City, Canada

Moineau and colleagues demonstrated that CRISPR-Cas9 creates double-stranded breaks in target DNA at precise positions, 3 nucleotides upstream of the PAM (Garneau et al., 2010). They also confirmed that Cas9 is the only protein required for cleavage in the CRISPR-Cas9 system. This is a distinguishing feature of Type II CRISPR systems, in which interference is mediated by a single large protein (here Cas9) in conjunction with crRNAs.

Discovery of tracrRNA for Cas9 system

March, 2011 — Emmanuelle Charpentier, Umea University, Sweden and University of Vienna, Austria

The final piece to the puzzle in the mechanism of natural CRISPR-Cas9-guided interference came from the group of Emmanuelle Charpentier. They performed small RNA sequencing on Streptococcus pyogenes, which has a Cas9-containing CRISPR-Cas system. They discovered that in addition to the crRNA, a second small RNA exists, which they called trans-activating CRISPR RNA (tracrRNA) (Deltcheva et al., 2011).  They showed that tracrRNA forms a duplex with crRNA, and that it is this duplex that guides Cas9 to its targets.

CRISPR systems can function heterologously in other species

July, 2011 — Virginijus Siksnys, Vilnius University, Lithuania

Siksnys and colleagues cloned the entire CRISPR-Cas locus from S. thermophilus (a Type II system) and expressed it in E. coli (which does not contain a Type II system), where they demonstrated that it was capable of providing plasmid resistance (Sapranauskas et al., 2011). This suggested that CRISPR systems are self-contained units and verified that all of the required components of the Type II system were known.

Biochemical characterization of Cas9-mediated cleavage

September, 2012 — Virginijus Siksnys, Vilnius University, Lithuania

Taking advantage of their heterologous system, Siksnys and his team purified Cas9 in complex with crRNA from the E. coli strain engineered to carry the S. thermophilus CRISPR locus and undertook a series of biochemical experiments to mechanistically characterize Cas9’s mode of action (Gasiunas et al., 2012).They verified the cleavage site and the requirement for the PAM, and using point mutations, they showed that the RuvC domain cleaves the non-complementary strand while the HNH domain cleaves the complementary site. They also noted that the crRNA could be trimmed down to a 20-nt stretch sufficient for efficient cleavage. Most impressively, they showed that they could reprogram Cas9 to a target a site of their choosing by changing the sequence of the crRNA.

June, 2012 — Charpentier and Jennifer Doudna, University of California, Berkeley

Similar findings as those in Gasiunas et al. were reported at almost the same time by Emmanuelle Charpentier in collaboration with Jennifer Doudna at the University of California, Berkeley (Jinek et al., 2012). Charpentier and Doudna also reported that the crRNA and the tracrRNA could be fused together to create a single, synthetic guide, further simplifying the system. (Although published in June 2012, this paper was submitted after Gasiunas et al.)

CRISPR-Cas9 harnessed for genome editing

January, 2013 — Feng Zhang, Broad Institute of MIT and Harvard, McGovern Institute for Brain Research at MIT, Massachusetts

Zhang, who had previously worked on other genome editing systems such as TALENs, was first to successfully adapt CRISPR-Cas9 for genome editing in eukaryotic cells (Cong et al., 2013). Zhang and his team engineered two different Cas9 orthologs (from S. thermophilus and S. pyogenes) and demonstrated targeted genome cleavage in human and mouse cells. They also showed that the system (i) could be programmed to target multiple genomic loci, and (ii) could drive homology-directed repair. Researchers from George Church’s lab at Harvard University reported similar findings in the same issue of Science (Mali et al., 2013).

( Source: Broad Institute of MIT and Harvard)

DNA Sequencing

Since the completion of the Human Genome Project, technological improvements and automation have increased speed and lowered costs to the point where individual genes can be sequenced routinely, and some labs can sequence well over 100,000 billion bases per year, and an entire genome can be sequenced for just a few thousand dollars.

Many of these new technologies were developed with support from the National Human Genome Research Institute (NHGRI) Genome Technology Program and its Advanced DNA Sequencing Technology awards. One of NHGRI’s goals is to promote new technologies that could eventually reduce the cost of sequencing a human genome of even higher quality than is possible today and for less than $1,000.

Researchers now are able to compare large stretches of DNA – 1 million bases or more – from different individuals quickly and cheaply. Such comparisons can yield an enormous amount of information about the role of inheritance in susceptibility to disease and in response to environmental influences. In addition, the ability to sequence the genome more rapidly and cost-effectively creates vast potential for diagnostics and therapies.

Although routine DNA sequencing in the doctor’s office is still many years away, some large medical centers have begun to use sequencing to detect and treat some diseases. In cancer, for example, physicians are increasingly able to use sequence data to identify the particular type of cancer a patient has. This enables the physician to make better choices for treatments.

Researchers in the NHGRI-supported Undiagnosed Diseases Program use DNA sequencing to try to identify the genetic causes of rare diseases. Other researchers are studying its use in screening newborns for disease and disease risk.

Moreover, The Cancer Genome Atlas project, which is supported by NHGRI and the National Cancer Institute, is using DNA sequencing to unravel the genomic details of some 30 cancer types.  Another National Institutes of Health program examines how gene activity is controlled in different tissues and the role of gene regulation in disease. Ongoing and planned large-scale projects use DNA sequencing to examine the development of common and complex diseases, such as heart disease and diabetes, and in inherited diseases that cause physical malformations, developmental delay and metabolic diseases.

(Resource: National Human Genome Research Institute)

Genomics Market Worth $19.0 Billion by 2018

According to a new market research report published by MarketsandMarkets, the global Genomics Market earned revenues of $11.11 Billion in 2013 and estimates this market to reach over $19.0 Billion by 2018.

The global Genomics Market was dominated by Tier I players such as Roche Diagnostics, Life Technologies, QIAGEN, Illumina, and Bio-Rad. Roche Diagnostics was the market leader in the global genomics market in 2013. This may be attributed to its strategic integration with NimbleGen’s MS 200 microarray scanner, as well as its co-promotional agreement with SoftGenetics in 2012. Significant Tier II players include BGI, PerkinElmer, and Hologic Gen-Probe, and Tier III companies included Oxford Gene Technology, Knome, and Phalanx Biotech. Notable tier IV players in this market were LGC Genomics and DNA STAR.

The Global Genomics market was found to be increasingly consolidated, owing to various restructuring activities in the past five years. Tier I and Tier II companies were the key acquirers, while Tier III companies were found to be the targets of M&A activities. Besides mergers and acquisitions, alliances were also a prominent strategy amongst leading players. Technology licensing was the most prevalent motive of such alliances. The acquisition of Life Technologies by Thermo Fischer in 2014 is expected to significantly change the competitive landscape of the global genomics market, as two key players in this market merge to consolidate Thermo Fischer’s market shares in coming years. The Instrument and Consumables product segments were dominated by companies with a global presence, while the Services market was dominated by small to medium-scale service providers with a regional presence.

Based on technology, the PCR technology segment accounted for the largest revenue share of the global genomics technology market in 2013. However, the DNA sequencing technology segment is expected to grow significantly during the forecast period. Market adoption of silica-based technology and magnetic bead technology was highest in the nucleic acid extraction and purification technology segment. The fast spin technology was found to be the most promising upcoming technology, as it offers improved clinical efficiency, and reduced time and cost. Other technology sub-segments expected to witness high rates of adoption include optical microarrays, micro fluidic systems, sequencing by synthesis (SBS), and single-molecule real-time (SMRT).

North America accounted for the highest share of revenues from the global Genomics Market, owing to the region’s focus on genomics research to improve human health. The APAC region is expected to witness the highest CAGR during the forecast period, owing to the increasing awareness of genomics applications in this region. Notable emerging regional markets for genomics include India, China, South Korea, Mexico, Brazil, Russia, and Turkey.

( Resource: MarketsandMarkets)