Leaders in Pharmaceutical Business Intelligence Group, LLC, Doing Business As LPBI Group, Newton, MA

Delineating a Role for CRISPR-Cas9 in Pharmaceutical Targeting

Author & Curator: Larry H. Bernstein, MD, FCAP

Chief Scientific Officer, Leaders in Pharmaceutical Intelligence, Boston, MA

http://pharmaceuticalintelligence.com

Correspondence:
larry.bernstein@gmail.com

Delineating a Role for CRISPR-Cas9 in Pharmaceutical Targeting, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

Abstract

The recent development of advanced methods for genome engineering has superceded methods already in used in recent years of the 21^st century.  The CRISPR-Cas9 application for genome editing has real potential for pharmaceutical development, and perhaps also for diagnostics.  The importance of conjoint development of diagnostics and therapeutics can’t be overstressed. Further, the limitations of the method have to be viewed in the light of the historical development of inborn errors of human metabolism, and current understanding of complex polygenomic and environmental risk factors.

Key words: classic model, CRISPR-Cas9, DNA, genome, genome editing, genetic diseases, Hardy-Weinberg equilibrium, inborn errors of metabolism, polygenetic diseases, RNA, RNA_i, translation.

Abbreviations: CRISPR-Cas9; DNA; HWE; RNA.

Introduction.

Genome editing technologies enable the deletion, insertion or correction of DNA at specific targeted sites within an organism’s genome. The power of the technology lies in its ability to specifically target any site in the genome and to alter the DNA sequence at that site. This has opened the door to potentially curing diseases caused by genetic defects, whether inherited or acquired.

Genome editing can be applied across many diverse fields of science. It has allowed researchers to gain a much deeper understanding of the role played by individual genes. Researchers working in the biomedical field use these techniques to address diseases that are known to have a genetic origin.

Early genome-editing research focused on the use of zinc finger nucleases and transcription activator-like effector nucleases (TALENs), which laid important foundations in establishing genome engineering as a potential approach for treating human diseases.

The recent discovery of CRISPR-Cas9, followed by work demonstrating its advantages over traditional approaches, promises a step-change in the use of genome editing to develop transformative medicines for serious human diseases.

Cas9* is an endonuclease (an enzyme) that can be easily programmed with RNA to cut DNA at targeted sites within the genome, enabling the deletion, insertion or correction of target genes, including those that cause diseases, with surgical precision. By using CRISPR-Cas9* genome-editing technology, scientists and clinicians are conducting pioneering research with a view to tackling both recessive and dominant genetic defects.

In order to find a place for CRISPR-Cas9 in gene therapy, it becomes necessary to consider inborn errors of metabolism and the evolution of traditional approaches to genetic diseases. Traditional gene therapy approaches to date have only been useful in correcting some recessive genetic disorders. Thanks to its ease of use and broad applicability, CRISPR-Cas9 has truly democratized genome editing and transformed many areas of research. Thousands of academic laboratories across the world are carrying out research using the technology. To this point, the technology known as CRISPR-Cas9 has been a science project, a research tool with enormous potential.

Genetic Disorders

A genetic disorder is a genetic problem caused by one or more abnormalities in the genome, especially a condition that is present from birth (congenital). Most genetic disorders are quite rare and affect one person in every several thousands or millions.

Genetic disorders may or may not be heritable, i.e., passed down from the parents’ genes. In non-heritable genetic disorders, defects may be caused by new mutations or changes to the DNA. In such cases, the defect will only be heritable if it occurs in the germ line. The same disease, such as some forms of cancer, may be caused by an inherited genetic condition in some people, by new mutations in other people, and mainly by environmental causes in still other people. Whether, when and to what extent a person with the genetic defect or abnormality will actually suffer from the disease is almost always affected by the environmental factors and events in the person’s development.

A single-gene disorder is the result of a single mutated gene. Over 4000 human diseases are caused by single-gene defects.^[4] Single-gene disorders can be passed on to subsequent generations in several ways. Genomic imprinting and uniparental disomy, however, may affect inheritance patterns. The divisions betweenrecessive and dominant types are not “hard and fast”, although the divisions between autosomal and X-linked types are (since the latter types are distinguished purely based on the chromosomal location of the gene). For example, achondroplasia is typically considered a dominant disorder, but children with two genes for achondroplasia have a severe skeletal disorder of which achondroplasics could be viewed as carriers. Sickle-cell anemia is also considered a recessive condition, but heterozygous carriers have increased resistance to malaria in early childhood, which could be described as a related dominant condition.^[5] When a couple where one partner or both are sufferers or carriers of a single-gene disorder wish to have a child, they can do so through in vitro fertilization, which means they can then have a preimplantation genetic diagnosis to check whether the embryo has the genetic disorder.^[6]

Heritable Diseases and Normal Variants

• Amino Acid Metabolism Disorders
• Carbohydrate Metabolism Disorders
• Congenital Muscle Diseases
• Connective Tissue, Muscle, and Bone Disorders
• Gonadal Dysgenesis Syndromes
• Hemoglobin Loci Mutations
• Heritable Cancer Syndromes and other Cancers
• Increased Chromosome Breakage Syndromes
• Lipid Metabolism Disorders
• Metal Metabolism Disorders
• Mitochondrial Genome Disorders
• Nervous System Disorders
• Nucleotide and Nucleic Acid Metabolism Disorders
• Ophthalmologic Disorders
• Other Known Biochemistry Disorders
• Repair Defective and Chromosome Instability Syndromes
• Steroid Metabolism Disorders
• Trinucleotide Expansion Disorders
• Uncertain Biochemical Etiology Disorders
• X Chromosome Marker Disorders

Identification of Genes for Childhood Heritable Diseases

Annual Review of Medicine Jan 2014; 65: 19-31

Boycott KM, Dyment DA, Sawyer SL, Vanstone MR, and Beaulieu CL.

Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, Ontario, K1H 8L1 Canada

http://dx.doi.org:/10.1146/annurev-med-101712-122108

Genes causing rare heritable childhood diseases are being discovered at an accelerating pace driven by the decreasing cost and increasing accessibility of next-generation DNA sequencing combined with the maturation of strategies for successful gene identification. The findings are shedding light on the biological mechanisms of childhood disease and broadening the phenotypic spectrum of many clinical syndromes. Still, thousands of childhood disease genes remain to be identified, and given their increasing rarity, this will require large-scale collaboration that includes mechanisms for sharing phenotypic and genotypic data sets. Nonetheless, genomic technologies are poised for widespread translation to clinical practice for the benefit of children and families living with these rare diseases.

Single gene defects result in abnormalities in the synthesis or catabolism of proteins, carbohydrates, fats, or complex molecules. Most are due to a defect in an enzyme or transport protein, which results in a block in a metabolic pathway. Effects are due to toxic accumulations of substrates before the block, intermediates from alternative metabolic pathways, defects in energy production and use caused by a deficiency of products beyond the block, or a combination of these metabolic deviations. Nearly every metabolic disease has several forms that vary in age of onset, clinical severity, and, often, mode of inheritance.

There is a large number of inborn errors of metabolism.

A few examples are:

Fructose intolerance
Galactosemia
Maple sugar urine disease (MSUD)
Phenylketonuria (PKU)

Newborn screening tests can identify some of these disorders

Categories of inborn errors of metabolism, or IEMs, are as follows:

• Disorders that result in toxic accumulation
  • Disorders of protein metabolism (eg, amino acidopathies, organic acidopathies, urea cycle defects)
  • Disorders of carbohydrate intolerance
  • Lysosomal storage disorders
• Disorders of energy production, utilization
  • Fatty acid oxidation defects
  • Disorders of carbohydrate utilization, production (ie, glycogen storage disorders, disorders of gluconeogenesis and glycogenolysis)
  • Mitochondrial disorders
  • Peroxisomal disorders

Giants in the 20^th century study of genetic medicine

1. Victor Almon McKusick

Victor Almon McKusick (October 21, 1921 – July 22, 2008), an internist and medical geneticist, was the University Professor of Medical Genetics and Professor of Medicine at the Johns Hopkins Hospital, Baltimore, MD, USA.^[1] He was a proponent of the mapping of the human genome due to its use for studying congenital diseases. He is well known for his studies of the Amish and, what he called, “little people”. He was the original author and, until his death, remained chief editor of Mendelian Inheritance in Man (MIM) and its online counterpart Online Mendelian Inheritance in Man (OMIM). He is widely known as the “father of medical genetics”.^[2]

McKusick traveled to Copenhagen to speak about the heritable disorders of connective tissue at the first international congress of human genetics. The meeting looms as the birthplace of the medical genetics field.^[2] In the following decades, McKusick created and chaired a new Division of Medical Genetics at Hopkins beginning in 1957. In 1973, he served as Physician-in-Chief, William Osler Professor of Medicine, and Chairman of the Department of Medicine at Johns Hopkins Hospital and School of Medicine.^[6]  He held concurrent appointments as University Professor of Medical Genetics at the McKusick–Nathans Institute of Genetic Medicine, Professor of Medicine at the Johns Hopkins School of Medicine, Professor of Epidemiology at the Johns Hopkins Bloomberg School of Public Health, and Professor of Biology at Johns Hopkins University.^[5] He co-founded Genomics in 1987 with Dr. Frank Ruddle, and served as an editor.^[6] He was a lead investigator in determining if Abraham Lincoln had Marfan syndrome.^[8]

2. Elizabeth F. Neufeld

Born in France, Elizabeth Neufeld immigrated to the United States in 1940. She obtained a BS from Queens College, New York and a Ph.D. from the University of California Berkeley. After postdoctoral training in, she moved to the NIH in Bethesda, MD, where she began her studies of a rare group of genetic diseases. She moved back to California in 1984 as Chair of the Department of Biological Chemistry – a position that she occupied till 2004.

The brain in a mouse model of a genetic lysosomal disorder, Sanfilippo syndrome type B

Our interests have long been the cause, consequences and treatment of human genetic diseases due to deficiency of lysosomal enzymes. The disease currently under investigation is the Sanfilippo syndrome type B (MPS III B). It is caused by mutation in the NAGLU gene, with resulting deficiency of the lysosomal enzyme alpha-N-acetyl-glucosaminidase and accumulation of its substrate (heparan sulfate). The disease manifests itself in childhood by severe mental retardation and intractable behavioral problems. The neurologic deterioration progresses to dementia, with death usually in the second decade. We use a mouse knockout model (Naglu -/-) in order to study the pathophysiology of the disease and to develop therapy. Because of the special cell biology of lysosomal enzymes, which can be taken up by receptor-mediated endocytosis, exogenous administration of the enzyme could theoretically cure the disease. Unfortunately, the blood-brain barrier (BBB) prevents the therapeutic enzyme from reaching the brain. Part of our current research is to develop a novel technology to get lysosomal enzymes across the BBB. We also study changes in gene and protein expression in some specific parts of the brain, in which there is accumulation of certain lipids and proteins which seem unrelated biochemically to each other or to the primary defect. We try to understand the cause and consequences of these accumulations. Although they are secondary defects, they may be relevant to the pathophysiology of the dieease and may have represent targets for pharmacologic intervention.

Neufeld began her scientific studies at a time when few women chose science as a career. The historical bias against women in science, compounded with an influx of men coming back from the Second World War and going to college, made positions for women rare; few women could be found in the science faculties of colleges and universities.

When she first began working on Hurler syndrome in 1967, she initially thought the problem might stem from faulty regulation of the sugars, but experiments showed the problem was in fact the abnormally slow rate at which the sugars were broken down. Working with fellow scientist Joseph Fratantoni, Neufeld attempted to isolate the problem by tagging mucopolysaccharides with radioactive sulfate, as well as mixing normal cells with MPS patient cells. Fratantoni inadvertently mixed cells from a Hurler patient and a Hunter patient—and the result was a nearly normal cell culture. The two cultures had essentially “cured” each other.

In 1973 Neufeld was named chief of NIH’s Section of Human Biochemical Genetics, and in 1979 she was named chief of the Genetics and Biochemistry Branch of the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases (NIADDK). She served as deputy director in NIADDK’s Division of Intramural Research from 1981 to 1983. In 1984 Neufeld went back to the University of California, this time the Los Angeles campus, as chair of the biological chemistry department.

Neufeld’s research opened the way for prenatal diagnosis of such life-threatening fetal disorders as Hurler syndrome. Neufeld chaired the Scientific Advisory Board of the National MPS Society and was president of the American Society for Biochemistry and Molecular Biology from 1992 to 1993. She was elected to both the National Academy of Sciences (USA) and the American Academy of Arts and Sciences in 1977 and named a fellow of the American Association for Advancement in Science in 1988. In 1990 she was named California Scientist of the Year. She was awarded the Wolf Prize, the Albert Lasker Award for Clinical Medical Research, and was awarded the National Medal of Science in 1994 “for her contributions to the understanding of the lysosomal storage diseases, demonstrating the strong linkage between basic and applied scientific investigation.”^[3]

3. Jarvis “Jay” Edwin Seegmiller, M.D.

“Jay Seegmiller was one of the giants of American medicine,” said Edward Holmes, M.D., Vice Chancellor of Health Sciences and dean of the School of Medicine at UCSD. “He and his trainees have made innumerable contributions to our understanding of the pathogenesis of many human disorders. Seegmiller was one of the country’s leading researchers in intermediary metabolism, with a focus on purine metabolism and inherited metabolism.  He worked in the field of human biochemical genetics, with a special interest in the mechanisms by which genetically determined defects of metabolism lead to various forms of arthritis.  His laboratory identified a wide range of primary metabolic defects in metabolism responsible for development of gout.

He is perhaps best known for his discovery of the enzyme defect in Lesch-Nyhan Syndrome, a fatal disorder of the nervous system causing severe mental retardation and self-mutilation impulses.  As Director of the Human Biochemical Genetics Program at UCSD, Seegmiller’s investigations into the translation of genetic research and methods of prevention, detection and treatment of hereditary diseases led to Congressional testimony on the possibility of controlling genetic disease in the United States.  As a result, genetic referral centers have been established throughout the country.

He joined the newly established UCSD School of Medicine in 1969 as head of the Arthritis Division of the Department of Medicine. There, he directed a research program in human biochemical genetics involving senior faculty from five departments within the School of Medicine.  While a professor at UCSD, he served as a Macy Scholar both at Oxford University and at the Basel Institute in Switzerland, as well as a Guggenheim Fellow at the Swiss Institute for Experimental Cancer Research in Lausanne.

In 1983, he became the founding director of what is today UCSD’s Stein Institute for Research on Aging (SIRA). Even after his retirement, he continued to serve as Associate Director of SIRA from 1990 until his death.

“He had the foresight of proposing the formation of and then establishing a new Institute on Aging at UCSD before there was any such Institute in the entire UC system,” said Dilip Jeste, M.D., the Estelle and Edgar Levi Chair in Aging, Professor of Psychiatry and Neurosciences and current Director of SIRA.   “He was himself a role model of successful aging, and continued working in the SIRA till his very last days.

Seegmiller received his Doctor of Medicine with honors from the University of Chicago in 1948.  After he completed his internship at Johns Hopkins Hospital in Baltimore, Maryland, he trained with Bernard Horecker of the National Institute of Arthritis and Metabolic Disease at the National Institutes of Health.

Seegmiller was appointed Senior Investigator of the National Institute of Arthritis and Metabolic Disease in 1954, where he carried out biochemical and clinical studies of human hereditary disease, with a special interest in those causing various forms of arthritis.  He became Assistant Scientific Director of the Institute in 1960, and was appointed Chief of the section on Human Biochemical Genetics in 1966, becoming one of several NIH leaders recruited to help launch UC San Diego’s new medical school.

Seegmiller’s clinical activities included studies in life longevity in South America.  In 1974, he joined a team of notable scientists and traveled to the remote village of Vilcabamba in Ecuador, to find out what role genetic factors played in the population of the Andean villagers who comprised some of the longest-living people in the world.  His later work led to the discovery of free radicals and their damaging effects in the human ability to withstand diseases, bringing forward new investigations on human aging at SIRA.

Seegmiller was a member of the National Academy of Sciences, the American Academy of Arts and Sciences, and was the recipient of numerous prizes and awards in honor of his extraordinary achievements in science and medicine.  He received the United States Public Health Distinguished Service Award in 1969; and was honored as Master of the American College of Rheumatology (ACR) in 1992. He was on the advisory boards for the National Genetics Foundation, the City of Hope Medical Center in Duarte, California, the Task Force on Endocrinology and Metabolism for NIH, the Executive Editorial Board for Analytical Biochemistry, and was President of the Western Association of Physicians in 1979.

What has changed?

The 21^st century has seen the mapping of the human genome. The huge focus on the genome came after the Watson and Crick discovery put the genome at the center of the translational network with the central hypothesis. What followed was transcription of RNA into placement of an amino acid into protein. The central hypothesis is DNA           RNA           protein.  However, RNA_i and non-translational RNA are now also important.  RNA has a role in suppressing translation, as do proteins by allosteric effects. In addition, the most common diseases involved in age related change are strongly responsive to extracellular matrix effects, ionic fluxes, effects on the cellular matrix, and involve multicentric genome expression. This mode of expression leads one to think hard about the therapeutic target, or targets. The effect of RNA or of protein interacting with the genome is not an element of the classic construct.

Identifying a part of the problem

Type 2 diabetes mellitus, hypertension, arrhythmias, atherosclerotic plaque development, cancer, congestive heart disease, pulmonary hypertension, pulmonary interstitial sclerosis, and renovascular disease are among the common diseases that develop during a lifetime. The phenotypic presentations may have genomic associations, and there may also be population variants.  There is also a cross-talk between these phenotypic expressions. Classically, medical terminology has been based on signs and symptoms of disease.  In the increasingly complex experience, the laboratory has played an increased role in the diagnosis as well as prognostication. The laboratory experience with respect to the practice of medicine has heavily relied of either proteins, enzymes, or the products of chemical reactions.  The use of genomic profiling has rapidly emerged in the laboratory armamentarium, but has had a slow ascent in practice.

Case in Point. Pompe’s disease

William Canfield is a glycobiologist, chief scientific officer and founder of an Oklahoma City-based biotechnology company, Novazyme, which was acquired by Genzyme in August 2001 and developed, among other things, an enzyme that can stabilize (but not cure) Pompe disease, based on Canfield’s ongoing research since 1998.^[1][2]   

John Crowley took over a position as a CEO in Novazyme after leaving Bristol-Myers Squibb in March 2000 and together with Dr. Y. T. Chen^[4] at Duke University pushed for expedited approval by the U.S. Food and Drug Administration (FDA) of a new drug compound, NZ-1001 under orphan drug designation for the treatment of Glycogen storage disease type II in October 2005. The FDA stated: “We have determined that Novazyme’s recombinant human highly phosphorylated acid alpha-glucosidase (rhHPGAA) qualifies for orphan designation for enzyme replacement therapy in patients with all subtypes of glycogen storage disease type II (Pompe’s disease).” ^[5][6] Subsequent research at Genzyme on NZ-1001 along with three other potential compounds brought approval of the first enzyme replacement therapy for Pompe’s disease – Alglucosidase alfa (Myozyme or Lumizyme, Genzyme Inc) in 2006.^[7]

William Canfields work with Pompes Disease was fictionalized and made the subject of a 2010 movie Extraordinary Measures in which he is called Dr. Robert Stonehill and played by Harrison Ford.^[8]

Case in point.  Polymorphisms in the long non-coding RNA

Hypertension (HT) is a complex disorder influenced by both genetic and environmental factors. Recent genome-wide association studies have identified a major risk locus for atherosclerosis on chromosome 9p21.3 (chr9p21.3). SNPs within the coding sequences of CDKN2A/B proteins and the long non-coding RNA CDKN2B-AS1 could potentially contribute to HT development. Such a study has now been done. The findings suggest that SNPs rs10757274, rs2383207, rs10757278, and rs1333049, particularly those within the CDKN2B-AS1 gene, and related haplotypes may confer increased susceptibility to HT development. (unpublished)

Case in point. Lipoprotein Lipase and Atherosclerosis

Lipoprotein lipase (LPL) plays a pivotal role in lipids and metabolism of lipoprotein. Dysfunctions of LPL have been found to be associated with dyslipidemia, obesity and insulin resistance.Dyslipidemia, obesity and insulin resistance are risk factor of atherosclerosis. Japanese investigators have  hypothesized that elevating LPL activity would cause protection of atherosclerosis. (unpublished).

Case in  point. Holocaust survivors pass on stress.

Descendants of Holocaust Survivors Have Altered Stress Hormones

Case in point. Genome engineering with CRISPR-Cas9

The new frontier of genome engineering with CRISPR-Cas9

GENOME EDITING

Jennifer A. Doudna* and Emmanuelle Charpentier*
Science Nov 2014; 346(6213) 1258096:1077 – 1087.
http://dx.doi.org:/10.1126/science.1258096

BACKGROUND: Technologies for making and manipulating DNA have enabled advances in biology ever since the discovery of the DNA double helix. But introducing site-specific modifications in the genomes of cells and organisms remained elusive. Early approaches relied on the principle of site-specific recognition of DNA sequences by oligonucleotides, small molecules, or self-splicing introns. More recently, the site-directed zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs) using the principles of DNAprotein recognition were developed. However, difficulties of protein design, synthesis, and validation remained a barrier to

SUMMARY

The field of biology is now experiencing a transformative phase with the advent of facile genome engineering in animals and plants using RNA-programmable CRISPR-Cas9. The CRISPR-Cas9 technology originates from type II CRISPR-Cas systems, which provide bacteria with adaptive immunity to viruses and plasmids. The CRISPR associated protein Cas9 is an endonuclease that uses a guide sequence within an RNA duplex, tracrRNA:crRNA, to form base pairs with DNA target sequences, enabling Cas9 to introduce a site-specific double-strand break in the DNA. The dual tracrRNA:crRNA was engineered as a single guide RNA (sgRNA) that retains two critical features: a sequence at the 5  side that determines the DNA target site by Watson-Crick base-pairing and a duplex RNA structure at the 3 side that binds to Cas9. This finding created a simple two-component system in which changes in the guide sequence of the sgRNA program Cas9 to target any DNA sequence of interest. The simplicity of CRISPR-Cas9 programming, together with a unique DNA cleaving mechanism, the capacity for multiplexed target recognition, and the existence of many natural type II CRISPR-Cas system variants, has enabled remarkable developments using this cost-effective and easy-to-use technology to precisely and efficiently target, edit, modify, regulate, and mark genomic loci of a wide array of cells and organisms.

Figure (not shown)

The Cas9 enzyme (blue) generates breaks in double-stranded DNA by using its two catalytic centers (blades) to cleave each strand of a DNA target site (gold) next to a PAM sequence (red) and matching the 20-nucleotide sequence (orange) of the single guide RNA (sgRNA). The sgRNA includes a dual-RNA sequence derived from CRISPR RNA (light green) and a separate transcript (tracrRNA, dark green) that binds and stabilizes the Cas9 protein. Cas9-sgRNA–mediated DNA cleavage produces a blunt double-stranded break that triggers repair enzymes to disrupt or replace DNA sequences at or near the cleavage site. Catalytically inactive forms of Cas9 can also be used for programmable regulation of transcription and visualization of genomic loci.

This Review illustrates the power of the technology to systematically analyze gene functions in mammalian cells, study genomic rearrangements and the progression of cancers or other diseases, and potentially correct genetic mutations responsible for inherited disorders. CRISPR-Cas9 is having a major impact on functional genomics conducted in experimental systems. Its application in genome-wide studies will enable large-scale screening for drug targets and other phenotypes and will facilitate the generation of engineered animal models that will benefit pharmacological studies and the understanding of human diseases. CRISPR-Cas9 applications in plants and fungi also promise to change the pace and course of agricultural research. Future research directions to improve the technology will include engineering or identifying smaller Cas9 variants with distinct specificity that may be more amenable to delivery in human cells. Understanding the homology-directed repair mechanisms that follow Cas9-mediated DNA cleavage will enhance insertion of new or corrected sequences into genomes. The development of specific methods for efficient and safe delivery of Cas9 and its guide RNAs to cells and tissues will also be critical for applications of the technology in human gene therapy.

Case in point.

ZFN, TALEN and CRISPR/Cas-based methods for genome engineering

Thomas Gaj1,2,3, Charles A. Gersbach4,5, and Carlos F. Barbas III1,2,3 1The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, USA 2Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA 3Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA 4Department of Biomedical Engineering, Duke University, Durham, NC, USA 5Institutes for Genome Sciences and Policy, Duke University, Durham, NC, USA

Trends Biotechnol . 2013 July ; 31(7): 397–405. http://dx.doi.org:/10.1016/j.tibtech.2013.04.004

Abstract Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) comprise a powerful class of tools that are redefining the boundaries of biological research. These chimeric nucleases are composed of programmable, sequence-specific DNA-binding modules linked to a non-specific DNA cleavage domain. ZFNs and TALENs enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone nonhomologous end joining or homology-directed repair at specific genomic locations. Here, we review achievements made possible by site-specific nuclease technologies and discuss applications of these reagents for genetic analysis and manipulation. In addition, we highlight the therapeutic potential of ZFNs and TALENs and discuss future prospects for the field, including the emergence of CRISPR/Cas-based RNA-guided DNA endonucleases.

Keywords zinc-finger; TALE; CRISPR; nuclease; genome engineering

Classical and contemporary approaches for establishing gene function With the development of new and affordable methods for whole-genome sequencing, and the design and implementation of large-scale genome annotation projects, scientists’ are poised to deliver upon the promises of the Genomic Revolution to transform basic science and personalized medicine. The resulting wealth of information presents researchers with a new primary challenge of converting this enormous amount of data into functionally and clinically relevant knowledge. Central to this problem is the need for efficient and reliable methods that enable investigators to determine how genotype influences phenotype. Targeted gene inactivation via homologous recombination is a powerful method capable of providing conclusive information for evaluating gene function.

Several factors impede the use of these methods:

• the low efficiency at which engineered constructs are correctly inserted into the chromosomal target site,
• the need for time-consuming and labor-insensitive selection/screening strategies, and
• the potential for adverse mutagenic effects.

Targeted gene knockdown by RNA interference (RNAi) has provided researchers with a rapid, inexpensive and high-throughput alternative to homologous recombination. However, knockdown by RNAi is incomplete, varies between experiments and laboratories, has unpredictable off-target effects, and provides only temporary inhibition of gene function. These restrictions impede researchers’ ability to directly link phenotype to genotype and limit the practical application of RNAi technology.

In the past decade, a new approach has emerged that enables investigators to directly manipulate virtually any gene in a diverse range of cell types and organisms. This core technology – commonly referred to as “genome editing” – is based on the use of engineered nucleases composed of sequence-specific DNA-binding domains fused to a non-specific DNA cleavage module. These chimeric nucleases enable efficient and precise genetic modifications by inducing targeted DNA double-strand breaks (DSBs) that stimulate the cellular DNA repair mechanisms, including error-prone non-homologous end joining (NHEJ) and homology-directed repair (HDR).

Case in point.

CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology

The development of efficient and reliable ways to make precise, targeted changes to the genome of living cells is a long-standing goal for biomedical researchers. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement. This follows several attempts over the years to manipulate gene function, including homologous recombination and RNA interference (RNAi).

RNAi, in particular, became a laboratory staple enabling inexpensive and high-throughput interrogation of gene function, but it is hampered by providing only temporary inhibition of gene function and unpredictable off-target effects. Other recent approaches to targeted genome modification – zinc-finger nucleases (ZFNs), and transcription-activator like effector nucleases (TALENs) – enable researchers to generate permanent mutations by introducing double stranded breaks to activate repair pathways. These approaches are costly and time-consuming to engineer, limiting their widespread use, particularly for large scale, high-throughput studies.

The Biology of Cas9

The functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. These repeats were initially discovered in the 1980s in E. coli, but their function wasn’t confirmed until 2007 by Barrangou and colleagues, who demonstrated that S. thermophilus can acquire resistance against a bacteriophage by integrating a genome fragment of an infectious virus into its CRISPR locus.

Three types of CRISPR mechanisms have been identified, of which type II has been the most studied. In this case, invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus amidst a series of short repeats (around 20 bps). The loci are transcribed, and transcripts are then processed to generate small RNAs (crRNA – CRISPR RNA), which are used to guide effector endonucleases that target invading DNA based on sequence complementarity (Figure 1) (not shown).

In the acquisition phase, foreign DNA is incorporated into the bacterial genome at the CRISPR loci. CRISPR loci is then transcribed and processed into crRNA during crRNA biogenesis. During interference, Cas9 endonuclease complexed with a crRNA and separate tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary sequence adjacent to the PAM sequence.

Investment in CRISPR technology

CRISPR Therapeutics is a biopharmaceutical company created to translate CRISPR-Cas9, a breakthrough genome-editing technology, into transformative medicines for serious human diseases. We are uniquely positioned to translate CRISPR-Cas9 technology into human therapeutics, thanks to its multi-disciplinary team of world-renowned academics, clinicians and drug developers.

CRISPR Therapeutics’ vision is to cure serious human diseases at the molecular level using CRISPR-Cas9. The company is headquartered in Basel, Switzerland and has operations in London, UK and Cambridge, Massachusetts.

The biopharmaceutical company that is focused on translating CRISPR-Cas9 gene-editing technology into transformative medicines for serious human diseases, congratulates its scientific founder, Dr. Emmanuelle Charpentier, for being named to TIME Magazine’s TIME 100 Most Influential People in the World alongside fellow CRISPR-Cas9 discoverer, Dr. Jennifer Doudna. In addition, Dr. Emmanuelle was awarded the Louis Jeantet Prize for Medicine, considered the most prestigious European award for researchers in the life sciences, for her discovery of the CRISPR-Cas9 gene editing tool. She will receive the award in a ceremony in Geneva, Switzerland, on April 22, 2015.

Dr. Charpentier has received numerous additional awards for her research, including in 2014 the Alexander von Humboldt Professorship, the Dr Paul Janssen Award, the Grand-Prix Jean-Pierre Lecocq (French Academy of Sciences), the Göran Gustafsson Prize (Royal Swedish Academy of Sciences) and in 2015 the Breakthrough Prize in Life Sciences. She was also selected as one of the American Foreign Policy magazine’s 100 Leading Global Thinkers for 2014.

Cambridge-based Editas Medicine announced a $120 million Series B round led by Bill Gates’s chief advisor for science and technology, Boris Nikolic. The list of financiers teaming with Nikolic reads like a rolodex of so-called crossover investors. Nikolic, who joined Editas’ board, made the investment through what’s been called “bng0,” a new U.S.-based investment company backed by “large family offices with a global presence and long-term investment horizon” and formed specifically to invest in Editas. CEO Katrine Bosley confirmed that Gates is one of the individuals investing in Editas alongside Nikolic. Editas has become the first of the group not only to attract crossover backers, but to begin discussing the diseases that its targeting.

Caribou Biosciences, one of the biotech startups working to advance a much-watched new technology for precise gene editing, has raised an $11 million Series A round from venture capital firms and Swiss drug giant Novartis.

The money will help Berkeley, CA-based Caribou speed up its efforts to adapt a versatile genome editing technique co-discovered by one of its founders, UC Berkeley professor Jennifer Doudna, for a range of uses, including drug research and development, and industrial technology.

Doudna and her collaborator, Emmanuelle Charpentier of the Helmholtz Center for Infection Research in Braunschweig, Germany, and Umeå University in Sweden, figured out how to transform a bacterial defense against viral infection into a tool to edit out abnormal sections of genes, such as those that cause hereditary diseases.

Caribou’s gene editing platform is based on two elements of that bacterial molecular machinery: a guiding mechanism called CRISPR (clustered, regularly interspaced palindromic repeats), and an enzyme called Cas9, or CRISPR-associated protein 9, molecular scissors that cut a segment of DNA. Caribou was founded in 2011 to commercialize the work from Doudna’s lab.

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