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CRISPR/Cas9 gene editing in hematology
ing of fusion proteins in hematopoietic cells was previous- ly accomplished by viral expression of fusion protein cDNA cloned from patients or by genomic engineering of mouse DNA to create chromosomal rearrangements using recombination systems (e.g., Cre-loxP), which is compli- cated.33 These aforementioned studies illustrate that CRISPR/Cas9 technology is a reliable and accurate approach to recreate chromosomal translocations, albeit at low efficiencies, providing a powerful tool for cancer stud- ies.
Another application of CRISPR/Cas9 technology that holds great promise is in the arena of functional genomics, in which it has been employed in genome-wide, loss-of- function screens in mammalian cells. Typically, lentiviral gRNA libraries are used in genetic screens for positive and negative selection,34,35 which have advantages over RNA interference-based screening with inherent incomplete gene knockdown. Another advantage of CRISPR/Cas9 is that it can target non-coding genomic regions, including promoters, enhancer elements, and intergenic regions. Positive selection studies screen for perturbations confer- ring enhanced self-renewal/proliferation/survival poten- tial to the interrogated cells, resulting in cell enrichment over time. By contrast, negative selection studies aim to identify genes essential for survival/proliferation that, when targeted, will cause cell depletion over time. In the context of myeloid malignancies, several high-throughput screens have been performed in drug target discovery applications. Using CRISPR/Cas9 to edit protein domains, Shi et al. identified cancer drug targets by screening 192 chromatin regulatory domains in murine AML cells, vali- dating six known drug targets and also revealing addition- al dependencies.36 In a study aiming to examine mecha- nisms of cytarabine drug resistance in AML cell lines, CRISPR/Cas9-based screening identified the deoxycyti- dine kinase gene as the primary contributor to cytarabine resistance.37 In addition to genome-wide CRISPR screens, targeted panel-based screens of previously selected genes would also allow the interrogation of biological processes, for example, cytokine signal transduction, cancer progres- sion or cell migration, which are suspected to be linked to a disease.
Generating mouse models using the CRISPR/Cas9 system
In vivo mouse models, usually generated by homolo- gous recombination strategies, have been instrumental in deciphering the role of point mutations, translocations, and DNA sequence indels in the context of a whole organism. CRISPR/Cas9 technology can be used to build both germline (heritable) and somatic mouse models in a fast and precise manner.38
Germline CRISPR/Cas9 mouse models
CRISPR/Cas9 has been employed to disrupt the splic- ing factor ZRSR1 in murine zygotes, resulting in altered erythrocyte function in adult mice, suggesting that ZRSR1-associated minor splicing could have an important role in terminal erythropoiesis.39 More recently, the tech- nology was used for the generation of novel hemophilia mouse models on an immunodeficient NSG (NOD/SCID/IL-2γ−/−) background.40 Hemophilia A and B are congenital, X-linked bleeding disorders caused by mutations in the genes encoding for the blood clotting factor VIII (F8) and factor IX (F9), respectively.
CRISPR/Cas9 and gRNA were microinjected into NSG mouse zygotes to generate mice with hemophilia A or hemophilia B. These models should allow the evaluation of the efficacy and safety of novel gene therapy vectors in hemophilia.
Given the importance of reporter mouse lines in bio- medical research, it is not surprising that CRISPR/Cas9 technology has been applied in the study of early devel- opmental processes. Recently, a knock-in mouse strain was created for dynamic tracking and enrichment of the MEIS1 transcription factor during hematopoiesis.41 This GFP-HA epitope tag reporter strategy and CRISPR/Cas9 gene editing could be employed to develop new reporter mouse lines to study other transcription factors important for hematopoiesis.
Mice carrying mutations in multiple genes have tradi- tionally been generated by sequential recombination in embryonic stem cells and/or intercrossing of mice with single mutations. CRISPR/Cas9 technology allows the generation of mice bearing different gene mutations in a more affordable, less labor-intensive and time-consuming manner than traditional approaches. Similar to the hemo- philia models describe above, mice with bi-allelic muta- tions in TET1 and TET2 were created by co-injection of targeting gRNA into mouse zygotes, which is a much faster approach compared with traditional techniques and allows one-step generation of animals with precise mutations.42 Accordingly, targeting multiple genes using CRISPR/Cas9 should facilitate, for example, the in vivo study of a family of genes with redundant functions. Indeed, Cas9 mRNA and multiple gRNA targeting B2M, IL2RG, PRF1, PRKDC, and RAG1 genes were microinject- ed together into mouse embryos to produce different immunodeficient mouse strains,43 thus generating new valuable tools to advance research in human HSPC xeno- transplantation.
There is increasing evidence that the acquisition of somatic mutations in HSC, leading to clonal hematopoiesis, is a cardiovascular risk factor. Indeed, DNMT3A and TET2 somatic mutations are drivers of clonal hematopoiesis of indeterminate potential, a state that predisposes to subsequent development of a hema- tologic malignancy or cardiovascular death.44 This recent study used CRISPR/Cas9 to inactivate DNMT3A and TET2 genes in HSPC and showed that atherosclerotic plaque size was markedly increased in reconstituted mice.45
Somatic CRISPR/Cas9 mouse models
Mouse models with somatic genome editing can be built by CRISPR/Cas9 modification of ex vivo cells fol- lowed by transplantation (murine cells) or xenotransplan- tation (human cells). For instance, the ability to modulate CRISPR/Cas9 activity has been exploited to perform doxycycline CRISPR/Cas9-inducible Trp53-knock- out/mutation. When HSPC isolated from a lymphoma transgenic model (Em-Myc) were transplanted, this resulted in accelerated lymphoma development in vivo. Thus, a highly efficient inducible CRISPR/Cas9 vector system can be used to identify novel gene mutations that drive tumorigenesis or to knock-out essential genes that are required for cell survival in vitro.46
As mentioned in the previous section, one of the unique features of the CRISPR/Cas9 system is its simplic- ity in enabling simultaneous disruption of several sites in
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