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Novel Genome-Editing Tools to Model and Correct Primary Immunodeficiencies.

Ott de Bruin LM, Volpi S, Musunuru K - Front Immunol (2015)

Bottom Line: With this treatment, severe complications may result due to integration within oncogenes.With these genome-editing tools a correct copy can be inserted in a precisely targeted "safe harbor." They can also be used to correct pathogenic mutations in situ and to develop cellular or animal models needed to study the pathogenic effects of specific genetic defects found in immunodeficient patients.This review discusses the advantages and disadvantages of these endonucleases in gene correction and modeling with an emphasis on CRISPR/Cas9, which offers the most promise due to its efficacy and versatility.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Boston Children's Hospital, Harvard Medical School , Boston, MA , USA ; Department of Pediatric Immunology, Wilhelmina Children's Hospital, University Medical Center Utrecht , Utrecht , Netherlands.

ABSTRACT
Severe combined immunodeficiency (SCID) and other severe non-SCID primary immunodeficiencies (non-SCID PID) can be treated by allogeneic hematopoietic stem cell (HSC) transplantation, but when histocompatibility leukocyte antigen-matched donors are lacking, this can be a high-risk procedure. Correcting the patient's own HSCs with gene therapy offers an attractive alternative. Gene therapies currently being used in clinical settings insert a functional copy of the entire gene by means of a viral vector. With this treatment, severe complications may result due to integration within oncogenes. A promising alternative is the use of endonucleases such as ZFNs, TALENs, and CRISPR/Cas9 to introduce a double-stranded break in the DNA and thus induce homology-directed repair. With these genome-editing tools a correct copy can be inserted in a precisely targeted "safe harbor." They can also be used to correct pathogenic mutations in situ and to develop cellular or animal models needed to study the pathogenic effects of specific genetic defects found in immunodeficient patients. This review discusses the advantages and disadvantages of these endonucleases in gene correction and modeling with an emphasis on CRISPR/Cas9, which offers the most promise due to its efficacy and versatility.

No MeSH data available.


Related in: MedlinePlus

Schematic representation of zygote injection with CRISPR/Cas9. (A) Injection of gRNA and Cas9 will lead to indels that can lead to a frameshift and an early stop codon thereby creating Knockout (KO) mice. (B) Addition of a highly homologous DNA template containing a specific mutation will result in Knock-in (KI) mice, through the process of homology-directed repair (HDR). Reagents are injected in the cytoplasm of the zygote. Alternatively, these can be injected in the pronucleus of the zygote, but cytoplasmic microinjection is simpler and less toxic.
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Figure 4: Schematic representation of zygote injection with CRISPR/Cas9. (A) Injection of gRNA and Cas9 will lead to indels that can lead to a frameshift and an early stop codon thereby creating Knockout (KO) mice. (B) Addition of a highly homologous DNA template containing a specific mutation will result in Knock-in (KI) mice, through the process of homology-directed repair (HDR). Reagents are injected in the cytoplasm of the zygote. Alternatively, these can be injected in the pronucleus of the zygote, but cytoplasmic microinjection is simpler and less toxic.

Mentions: In recent years, many animal models have been successfully generated using ZFNs, TALENs, or CRISPR/Cas9. TALENs and CRISPR/Cas9 have been used to generate knockout Caenorhabditis elegans models by injecting the endonucleases into the gonads (103–105). Similarly, more complicated animal models can be generated by injecting the endonucleases in mRNA form directly in zygotes (Figure 4). In the case of CRISPR/Cas9, this means that both the Cas9 and gRNA in RNA form are injected. Knock-in models can be generated by adding a DNA template to the injection mix, usually in the form of a single-stranded DNA oligonucleotide. Zebrafish models have been generated by injecting ZFNs or TALENs or CRISPR/Cas9 directly into the zygote (106–109). This has been done in murine zygotes with ZFNs (110–112), TALENs (113, 114), and extremely efficiently with CRISPR/Cas9 (115–117). New mouse models can be generated in just a few weeks, instead of taking 1–2 years as in the conventional strategy. With CRISPR/Cas9, the specific gRNA needed for the injections can be generated in a simple one-day procedure (118). NSG mice have been efficiently generated in this way (119). In other studies, the IgM locus has been successfully knocked out in rats via the injection of ZFNs and TALENs directly into the zygotes (120, 121). Similarly, a rat model of X-SCID has been generated using ZFNs (122). The multiplexing capacity of CRISPR/Cas9 has allowed for multiple genes being knocked out simultaneously (123). Endonucleases have been used to generate knockout models in animals not previously amenable to efficient genetic modification: rabbits with IL2RG, RAG1, or RAG2 knockout (124–127); hamsters with STAT2 knockout (128); mutant pigs (129–131); and most impressively, monkeys with RAG1 knockout (132). These kinds of animal models will enable disease studies of unprecedented sophistication.


Novel Genome-Editing Tools to Model and Correct Primary Immunodeficiencies.

Ott de Bruin LM, Volpi S, Musunuru K - Front Immunol (2015)

Schematic representation of zygote injection with CRISPR/Cas9. (A) Injection of gRNA and Cas9 will lead to indels that can lead to a frameshift and an early stop codon thereby creating Knockout (KO) mice. (B) Addition of a highly homologous DNA template containing a specific mutation will result in Knock-in (KI) mice, through the process of homology-directed repair (HDR). Reagents are injected in the cytoplasm of the zygote. Alternatively, these can be injected in the pronucleus of the zygote, but cytoplasmic microinjection is simpler and less toxic.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440404&req=5

Figure 4: Schematic representation of zygote injection with CRISPR/Cas9. (A) Injection of gRNA and Cas9 will lead to indels that can lead to a frameshift and an early stop codon thereby creating Knockout (KO) mice. (B) Addition of a highly homologous DNA template containing a specific mutation will result in Knock-in (KI) mice, through the process of homology-directed repair (HDR). Reagents are injected in the cytoplasm of the zygote. Alternatively, these can be injected in the pronucleus of the zygote, but cytoplasmic microinjection is simpler and less toxic.
Mentions: In recent years, many animal models have been successfully generated using ZFNs, TALENs, or CRISPR/Cas9. TALENs and CRISPR/Cas9 have been used to generate knockout Caenorhabditis elegans models by injecting the endonucleases into the gonads (103–105). Similarly, more complicated animal models can be generated by injecting the endonucleases in mRNA form directly in zygotes (Figure 4). In the case of CRISPR/Cas9, this means that both the Cas9 and gRNA in RNA form are injected. Knock-in models can be generated by adding a DNA template to the injection mix, usually in the form of a single-stranded DNA oligonucleotide. Zebrafish models have been generated by injecting ZFNs or TALENs or CRISPR/Cas9 directly into the zygote (106–109). This has been done in murine zygotes with ZFNs (110–112), TALENs (113, 114), and extremely efficiently with CRISPR/Cas9 (115–117). New mouse models can be generated in just a few weeks, instead of taking 1–2 years as in the conventional strategy. With CRISPR/Cas9, the specific gRNA needed for the injections can be generated in a simple one-day procedure (118). NSG mice have been efficiently generated in this way (119). In other studies, the IgM locus has been successfully knocked out in rats via the injection of ZFNs and TALENs directly into the zygotes (120, 121). Similarly, a rat model of X-SCID has been generated using ZFNs (122). The multiplexing capacity of CRISPR/Cas9 has allowed for multiple genes being knocked out simultaneously (123). Endonucleases have been used to generate knockout models in animals not previously amenable to efficient genetic modification: rabbits with IL2RG, RAG1, or RAG2 knockout (124–127); hamsters with STAT2 knockout (128); mutant pigs (129–131); and most impressively, monkeys with RAG1 knockout (132). These kinds of animal models will enable disease studies of unprecedented sophistication.

Bottom Line: With this treatment, severe complications may result due to integration within oncogenes.With these genome-editing tools a correct copy can be inserted in a precisely targeted "safe harbor." They can also be used to correct pathogenic mutations in situ and to develop cellular or animal models needed to study the pathogenic effects of specific genetic defects found in immunodeficient patients.This review discusses the advantages and disadvantages of these endonucleases in gene correction and modeling with an emphasis on CRISPR/Cas9, which offers the most promise due to its efficacy and versatility.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Boston Children's Hospital, Harvard Medical School , Boston, MA , USA ; Department of Pediatric Immunology, Wilhelmina Children's Hospital, University Medical Center Utrecht , Utrecht , Netherlands.

ABSTRACT
Severe combined immunodeficiency (SCID) and other severe non-SCID primary immunodeficiencies (non-SCID PID) can be treated by allogeneic hematopoietic stem cell (HSC) transplantation, but when histocompatibility leukocyte antigen-matched donors are lacking, this can be a high-risk procedure. Correcting the patient's own HSCs with gene therapy offers an attractive alternative. Gene therapies currently being used in clinical settings insert a functional copy of the entire gene by means of a viral vector. With this treatment, severe complications may result due to integration within oncogenes. A promising alternative is the use of endonucleases such as ZFNs, TALENs, and CRISPR/Cas9 to introduce a double-stranded break in the DNA and thus induce homology-directed repair. With these genome-editing tools a correct copy can be inserted in a precisely targeted "safe harbor." They can also be used to correct pathogenic mutations in situ and to develop cellular or animal models needed to study the pathogenic effects of specific genetic defects found in immunodeficient patients. This review discusses the advantages and disadvantages of these endonucleases in gene correction and modeling with an emphasis on CRISPR/Cas9, which offers the most promise due to its efficacy and versatility.

No MeSH data available.


Related in: MedlinePlus