VIRUSES
Chapter 11 Chapter 11
2. TALENs (transcription activator like effector nu- cleases) are similar to zinc finger nucleases. They
also contain the catalytic domain of the FokI nuclease, but the DNA binding modules are dif- ferent. They are derived from a bacterial plant pathogen, and they are structurally unrelated to zinc fingers.
3. RNA-guided nucleases recognize their target DNA not through protein modules, but by base pair- ing involving a guide RNA. The commonly used CRISPR/Cas system is derived from an adaptive im- mune system of bacteria. The bacteria incorporate pieces of virus DNA into their genome, transcribe them into RNA, and use this RNA to guide the Cas endonuclease to the corresponding sequences in vi- ral DNA. This system has first been used on mam- malian cells in 2013 and is rapidly becoming the prime tool for genome editing. It requires only the Cas nuclease and a guide RNA (gRNA) of about 20 nucleotides that is complementary to the tar- geted DNA, fused to an RNA of about 80 nucleo- tides that is a structural component of the enzyme (Fig. 11.19, B).
This system requires no time-consuming protein engi- neering but only a custom-synthesized RNA to guide the Cas endonuclease to its target DNA. It is even possible to edit multiple sites in the genome simulta- neously by administering Cas with several different gRNAs. As with the other methods, donor DNA needs to be administered with the nuclease if gene repair is intended.
Nucleases and donor DNA need to be brought into the nucleus of the target cells. Genes encoding the nu- cleases can be brought into the cell on viral vectors that do not integrate into the host cell DNA but express the nuclease gene for a short time period only. Alternatively, either the nuclease itself or its mRNA can be brought into the cell with various methods. Cellular uptake of reagents is currently the main challenge in the field of genome editing.
A
B
Target DNA
Double-strand break
Double-strand break
DNA “repaired”
with mutation
Target DNA with mutation
Repaired DNA DNA synthesis,
ligation Strand resection Nuclease cleavage, donor DNA Nuclease cleavage
NHEJ
5′
5′ 5′ 5′
5′ 5′
3′
3′3′ 3′
3′
3′
Fig. 11.18 Use of designer nucleases for genome editing.
A, Gene knockout. The nuclease introduces a double- strand break in the coding sequence of the targeted gene.
Repair by nonhomologous end joining (NHEJ) introduces a small deletion that results, in most cases, in an
inactivating frameshift mutation. B, Gene repair. A donor DNA (blue) that is homologous to the targeted sequence is introduced into the cell in addition to the nuclease. Under these conditions, most cuts are repaired by homologous repair.
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ANTISENSE OLIGONUCLEOTIDES CAN BLOCK THE EXPRESSION OF ROGUE GENES
Instead of destroying an undesirable gene with a de- signer nuclease, it may be sufficient to prevent the translation of its mRNA. Antisense technology is a set
of methods that target the mRNA of undesirable genes.
It brings oligonucleotides into the cell that are comple- mentary to the mRNA, hybridize with it, and block its translation. If the oligonucleotide is DNA rather than RNA, it induces the cleavage of the mRNA by RNase H.
This cellular enzyme cleaves the RNA strand in a DNA- RNA hybrid. It otherwise participates in primer removal during DNA replication, and also takes part in the cell’s antivirus defenses.
Antisense agents must have a length of at least 18 to 20 nucleotides to achieve sufficient selectivity for their target sequence, and nuclease-resistant oligonucleotide analogs are commonly used. Fig. 11.20 shows some examples. As with designer nucleases and donor DNA, cellular uptake of these antisense oligonucleotides is not very efficient and is a main impediment to their widespread use.
One form of antisense technology exploits the nat- ural process of RNA interference, which is described in Chapter 7. In this case the antisense oligonucleotide is a small interfering RNA (siRNA): a short piece of double-stranded RNA with a length of 20 to 25 base pairs that is complementary to a sequence in the unde- sirable mRNA. One of the RNA strands becomes bound to the Ago2 protein in the RNA-induced silencing com- plex (RISC), which then cleaves the targeted mRNA (see Chapter 7). In theory, a single RNA molecule can direct the enzyme to destroy thousands of mRNA mol- ecules carrying the complementary sequence.
GENES CAN BE ALTERED IN ANIMALS
There are three ways of studying gene function in labo- ratory animals, usually mice:
1. Knockout is selective disruption of a normal gene in the germline. It produces knockout mice, whose phenotype reveals the biological function of the knocked-out gene.
A
Zinc fingers
Zinc fingers Nuclease
DNA
DNA
B
gRNA Cas9 Fok1
Fok1
Fig. 11.19 Methods of genome editing. A, Use of zinc finger nucleases. Each zinc finger recognizes a 3-base-pair sequence in the target DNA. The Fok1 catalytic domain is fused with the sequence of zinc fingers. The two Fok1 half-enzymes bind to the target DNA through their zinc fingers. They have to dimerize before they can cut the DNA (↓). B, Genome editing with the CRISPR/Cas system. The Cas9 enzyme contains a guide RNA (gRNA, purple) that base- pairs with one of the DNA strands (red). It has two nuclease domains for cutting the two strands of the DNA (↑).
CH3
–S P
O Base
O O
O O
CH3
H3C P
O Base
O O
O
P O
CH3
–O P
O Base
O F O
O O
Fig. 11.20 Structural modifications in experimentally used antisense oligonucleotides. These modifications are intended to make the oligonucleotides resistant to nucleases, facilitate their uptake into cells, or increase their affinity for their mRNA targets.
2. Knockin is the insertion of a gene that is not nor- mally present in the genome. It produces transgenic animals.
3. Knockdown is achieved by preventing the translation of an mRNA, using RNA interference and similar methods. It does not change the genome. Unlike the other methods, which are performed in the zygote or early embryo, gene knockdown is done in adult mice.
The genome remains unchanged, and the manipula- tion is reversible.
All three methods are used to create animal models of human diseases. In addition, transgenic animals are used in “pharming.” For example, cattle and sheep that se- crete human hormones, clotting factors, or other thera- peutic proteins in their milk have been produced.
To produce transgenic mice, the donor DNA consists of the entire transgene with flanking sequences that are homologous with the cellular DNA. Designer nuclease and donor DNA are brought into the zygote or cultured embryonic stem cells by injection or electroporation.
Three strategies can be used to produce transgenic mice and knockout mice (Fig. 11.21):
1. The reagents are injected into the oocyte. After fer- tilization in the test tube, the resulting embryo is tested for the presence of the desired genetic change.
Successfully modified embryos are placed into the uterus of a foster animal.
2. The foreign gene is engineered into cultured embry- onic stem cells, followed by injection of the engi- neered stem cells into an embryo at the blastocyst
DNA injected into oocyte
A
Enucleated oocyte Transgenic nucleus
B
C
Fertilization
Transgenic zygote
Foreign DNA introduced by electroporation
Incubation, implantation of embryo in foster mouse
Transgenic mouse
Selection of transgenic cells
Injection into blastocyst
Chimeric mouse
Implantation in foster mouse
+
Incubation, implantation in foster mouse
Transgenic
“zygote” Transgenic
mouse
Fig. 11.21 Three methods for the production of genetically modified animals. A, Foreign DNA is injected into the oocyte.
B, Cultured embryonic stem cells are genetically modified. These cells are injected in the inner cell mass of a developing embryo at the blastocyst stage to produce a chimeric animal. C, Reproductive cloning with the nucleus of a genetically modified embryonic stem cell.
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stage. Being totipotent, these stem cells can contrib- ute to all tissues of the developing embryo. After im- plantation in a foster animal, the embryo develops into a chimeric animal. If some of the engineered stem cells enter the germline, the genetic modifica- tion can be transmitted to the animal’s descendants.
3. The gene is engineered into embryonic stem cells whose nuclei are then transferred into enucleated oocytes. The cloned animals that are produced with this method have the genetic modification in all their cells.
TISSUE-SPECIFIC GENE EXPRESSION CAN BE ENGINEERED INTO ANIMALS
The tissue-specific expression of an artificially intro- duced gene is determined in large part by its promoter.
For example, a genetic engineer who wants to make humans capable of cellulose digestion could combine a cellulase gene from a snail or a fungus with a signal sequence and the promoter of the gene for trypsinogen
or some other pancreatic zymogen. After introduction of the gene into the germline, the cellulase would be se- creted by the pancreas.
Gene knockouts can be made tissue specific with the help of loxP sites. The loxP site is a 34–base-pair palin- dromic sequence that is recognized by the Cre recom- binase, an integrase enzyme from a bacteriophage. It acts somewhat like a spliceosome but with DNA rather than RNA as a substrate. Cre recombinase cuts out the DNA between two loxP sites and splices together the flanking DNA.
Fig. 11.22 shows a procedure that has been used to knock out the gene for the insulin receptor specifically in adipose tissue. The transgenic mice have exon 4 of the insulin receptor gene flanked with loxP sites. They also have the Cre gene under the control of a promoter that permits gene expression only in adipose tissue.
The Cre recombinase does no harm to the normal DNA in adipose cells, which does not contain any loxP sites. Only exon 4 of the insulin receptor gene is cut out of the genome.
Fig. 11.22 Strategy for eliminating the insulin receptor gene selectively in adipose tissue. A strain of mice is created with loxP sites ( ) flanking one of the exons (exon 4) of the insulin receptor gene. Another strain is created with the cre gene under the control of an adipose tissue selective promoter. When these two strains are crossed to create mice with both kinds of genetic modification, the Cre recombinase excises exon 4 only in adipose tissue.
Designer nuclease, homologous
recombination Designer nuclease,
homologous recombination Exon 4
Intron Intron Insulin
receptor
gene Junk DNA
Mouse with exon 4 flanked by loxP sites
Adipose- specific promoter
Crossing
Mouse with cre gene cre gene
Exon 4 excised in adipose tissue
of hybrid mouse
+
The resulting mice cannot make insulin receptors in adipose tissue. Although their adipose tissue cannot re- spond to insulin, they are not diabetic because the insu- lin receptor is intact in other tissues. These mice are very lean, and they live longer than normal laboratory mice.
A similar result can be achieved with an antisense gene whose RNA is complementary to the targeted mRNA, or an artificial gene whose RNA transcript is processed to a siRNA. For example, an antisense gene for the insulin receptor with an adipose tissue specific promoter would prevent the synthesis of the insulin re- ceptor in adipose tissue without destroying the insulin receptor gene.
HUMAN GERMLINE GENOME EDITING IS TECHNICALLY POSSIBLE
In theory, transgenic humans can be produced by using a designer nuclease and a donor DNA with the desired gene sequence. For example, two parents who both have sickle cell disease cannot have a healthy child. All their children will have sickle cell disease. Only germline engi- neering applied to the oocyte or zygote can convert one of the sickle cell alleles back to the normal allele and permit the birth of a healthy child. It is even possible to correct multiple genetic flaws in the oocyte by using an RNA-guided nuclease.
The main reason for not using germline engineer- ing at this time is that it is not safe. The available designer nucleases do sometimes cleave off-target, creating serious mutations including chromosome breakage and chromosomal rearrangements. Also, the efficiency of genome editing with presently available methods is low.
Human artificial chromosomes might be a safer way of making better people. These chromosomes contain centromeres, telomeres, and replication origins, along with splice sites for the insertion of gene cassettes. The desired genes are inserted, and the chromosome is in- jected into the nucleus of the oocyte or zygote during in vitro fertilization. Genes that people might wish to give to their children include the following:
1. Life-prolonging genes. Some genetic manipulations in animals, including the adipose-selective insulin re- ceptor knockout shown in Fig. 11.22, are known to prolong life.
2. Tumor suppressor genes. These are normal genes for DNA repair or negative controls on mitosis whose homozygous inactivation contributes to cancer. For example, transgenic mice with an extra copy of the tumor suppressor gene TP53 (see Chapter 19) have a substantially reduced cancer risk. Many tumor sup- pressor genes exist, and having one or two extra cop- ies of each could protect people from cancer.
3. Genes that antagonize age-related changes. For example, accumulation of β-amyloid in Alzheimer disease (see Chapter 2) might be preventable by
a brain-expressed antisense gene for the amyloid precursor protein.
Human artificial chromosomes can be equipped with loxP sites left and right of the centromere and a Cre gene controlled by a germline-specific promoter. In that case, the centromere will be cut out and the chromosome will be destroyed in the germline, preventing its transmission to the next generation. When deciding about their chil- dren’s genes, parents will certainly want to give them not their own outdated chromosome but the most recent model!
SUMMARY
Highly efficient methods are available for fragmenta- tion of DNA, enzymatic amplification, propagation of DNA in genetically engineered microorganisms, and DNA sequencing.
The most important applications of molecular ge- netic techniques in medicine are genotyping and diag- nosis of genetic diseases. Adults, infants, fetuses and embryos can be tested for chromosomal rearrange- ments, single-gene disorders, and predisposition to multifactorial diseases. Prenatal and preimplantation genetic diagnoses are possible, and whole populations can be screened for problematic genes. DNA microar- rays can be used to test for thousands of mutations and genetic polymorphisms in a single procedure, and even whole-genome sequencing is becoming affordable as a diagnostic procedure.
Therapeutic uses that are under investigation in- clude somatic cell gene therapy and RNA interference.
Germline gene modifications have not yet been at- tempted in humans, although whole armies of knock- out mice and transgenic mice populate the research laboratories, and pharmaceutical proteins from trans- genic animals are used routinely in medicine.
Further Reading
Beaudet, A. L. (2013). The utility of chromosomal microarray analysis in developmental and behavioral pediatrics. Child Development, 84(1), 121–132.
Canaan, A., DeFuria, J., Perelman, E., Schultz, V., Seay, M., Tuck, D., … Weissman, S. (2014). Extended lifespan and reduced adiposity in mice lacking the FAT10 gene.
Proceedings of the National Academy of Sciences of the United States of America, 111(14), 5313–5318.
Garibyan, L., & Avashia, N. (2013). Research techniques made simple: Polymerase chain reaction (PCR). Journal of Investigative Dermatology, 133(3), e6.
Ginn, S. L., Alexander, I. E., Edelstein, M. I., Abedi, M. R.,
& Wixon, J. (2013). Gene therapy clinical trials worldwide to 2012—an update. The Journal of Gene Medicine, 15(2), 65–77.
Hitzemann, R., Bottomly, D., Durakjian, P., Walter, N., Iancu, O., Searles, R., … McWeeney, S. (2013). Gene, behavior and next-generation RNA sequencing. Genes, Brain and Behavior, 12(1), 1–12.
185 DNA Technology
Humbert, O., Davis, L., & Maizels, N. (2012). Targeted gene therapies: Tools, applications, optimization. Critical Reviews in Biochemistry and Molecular Biology, 47(3), 264–281.
Ishino, S., & Ishino, Y. (2014). DNA polymerases as useful reagents for biotechnology—the history of developmental research in the field. Frontiers in Microbiology, 5, 465.
Kitzman, J. O., Snyder, M. W., Ventura, M., Lewis, A. P., Qiu, R., Simmons, L. E., & Shendure, J. (2012). Noninvasive whole- genome sequencing of a human fetus. Science Translational Medicine, 4. 137ra76.
Koboldt, D. C., Steinberg, K. M., Larson, D. E., Wilson, R. K.,
& Mardis, E. R. (2013). The next-generation sequencing revolution and its impact on genomics. Cell, 155(1), 27–38.
Kung, A., Monné, S., Bankowski, B., Coates, A., & Wells, D.
(2015). Validation of next-generation sequencing for com- prehensive chromosome screening of embryos. Reproductive Biomedicine Online, 31(6), 760–769.
Loenen, W. A., Dryden, D. T., Raleigh, E. A., Wilson, G. G.,
& Murray, N. E. (2014). Highlights of the DNA cutters:
A short history of the restriction enzymes. Nucleic Acids Research, 42(1), 3–19.
Ma, Z., Lee, R. W., Li, B., Kenney, P., Wang, Y., Erikson, J., … Lao, K. (2013). Isothermal amplification method for next- generation sequencing. Proceedings of the National Academy of Sciences of the United States of America, 110(35), 14320–14323.
Maggio, I., & Gonçalves, M.A.F.V. (2015). Genome editing at the crossroads of delivery, specificity, and fidelity. Trends in Biotechnology, 33(5), 280–291.
Uno, N., Kazuki, Y., & Oshimura, M. (2014). Toward gene and cell therapies employing human artificial chro- mosomes in conjunction with stem cells. Cloning &
Transgenesis, 3, 122.
van Dijk, E. L., Auger, H., Jaszczyszyn, Y., & Thermes, C.
(2014). Ten years of next-generation sequencing technol- ogy. Trends in Genetics, 30(9), 418–426.
van Duyne, G. D. (2014). Cre recombinase. Microbiology Spectrum, 3(1). MDNA3-0014-2014.
van El, C. G., Cornel, M. C., Borry, P., Hastings, R. J., Fellmann, F., & Hodgson, S.V., & ESHG Public and Professional Policy Committee. (2013). Whole-genome sequencing in health care. Recommendations of the European Society of Human Genetics. European Journal of Human Genetics, 21, 580–584.
Xiao-Jie, L., Hui-Ying, X., Zun-Ping, K., Jin-Lian, C., &
Li-Juan, J. (2015). CRISPR-Cas9: A new and promising player in gene therapy. Journal of Medical Genetics, 52(5), 289–296.
Yang, H., Wang, H., Shivalila, C. S., Cheng, A. W., Shi, L., &
Jaenisch, R. (2013). One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell, 154(6), 1370–1379.
QUESTIONS
1. To sequence a piece of DNA with the dideoxy