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E. González-Romero et al.
CRISPR/Cas9-mediated cleavage followed by HDR has been employed to introduce point mutations or gene frag- ments into specific loci using donor template DNA flanked by 3' and 5' sequences homologous to the target region. However, creating a knock-in allele by homolo- gous recombination of a targeting construct using embry- onic stem cells (which could be used to produce a mouse model) or by CRISPR/Cas9 is not so different in terms of cost and time.22 CRISPR/Cas9 gene editing can help to elucidate the role of patients’ mutations by generating cellular models carrying these lesions. Along this line, patients with myelodysplastic syndromes frequently have mutations in splicing genes such as the P95H muta- tion in serine/arginine factor 2 (SRSF2), which regulates pre-mRNA splicing. Zhang et al. developed an SRSF2/P95H cell line using CRISPR/Cas9-mediated HDR, which resulted in a gain-of-function phenotype and changed its RNA-binding preferences, producing splicing misregulation. This illustrates how a mutation associated with myelodysplastic syndromes alters splicing patterns, some of which are relevant for disease and have thera- peutic potential.23 In acute myeloid leukemia, driver mutations can also cause and/or maintain leukemia24 and precise AML models are needed to develop novel, target- ed therapies. For instance, the R140Q mutation in the Krebs cycle enzyme isocitrate dehydrogenase 2 (IDH2) endows cells with neomorphic enzyme activity, generat- ing an oncometabolite that interferes with epigenetic cell regulation and contributes to malignant transformation. To study the molecular and functional characteristics of this driver mutation, genome editing was used in K562 cells to introduce the IDH2/R140Q mutation.25 Cells car- rying this mutation recapitulated the genetic, epigenetic and functional changes seen in IDH2-mutated patients, offering a suitable model for drug testing.
In addition to modeling disease, CRISPR/Cas9 has been employed to correct mutations in disease-associated genes using single-stranded donor oligonucleotides as DNA donor templates for HDR. For example, a loss-of- function mutation in the Additional sex combs like 1 (ASXL1) gene, frequently mutated in myelodysplastic syndromes, chronic myelomonocytic leukemia, and AML was corrected in a chronic myeloid leukemia cell line.26 Similarly, AML blasts (precursor cells) containing the IDH2R140Q mutation were corrected to restore cell function to wild-type status.25 These results constitute a proof-of- concept that CRISPR/Cas9 gene correction of primary
hematopoietic cells is feasible. Beyond hematopoietic cells, CRISPR/Cas9 genome editing of human induced pluripotent stem cells (iPSC) has been used to correct dis- ease-relevant mutations. For example, correction of the HCLS1 associated protein X-1 (HAX1) gene by CRISPR/Cas9-mediated HDR reversed the severe con- genital neutropenia phenotype in patient-specific iPSC.27 This is important given that iPSC are excellent platforms to model disease and also hold promise for use in patient- specific, cell-based regenerative therapy. Accordingly, hematopoietic cells carrying a mutation could be isolated from the patient, reprogrammed to iPSC, edited, differen- tiated to hematopoietic stem cells (HSC) and re-intro- duced by autologous HSC transplantation. However, the capability of iPSC-derived HSC to reconstitute the blood system in the long-term remains a challenge for clinical translation.28
Most pre-clinical models of CRISPR/Cas9-based gene repair have been based on precise but relatively poorly efficient HDR. The greater efficiency of NHEJ-based mutation correction in the absence of donor template DNA has been used successfully to repair frame-shift mutations. For example, in a study on X-linked chronic granulomatous disease, which is caused by mutations in the cytochrome b-245 heavy chain (CYBB) gene,29 patient- specific CYBB point mutations were successfully repaired by NHEJ – the dominant DSB-repair pathway in hematopoietic stem and progenitor cells (HSPC) – with gene repair efficiency being between 18-25%. The authors of this study assumed that approximately one- third of NHEJ-mediated indels should re-establish the open reading frame disrupted by the disease mutation, leading to a complete or partial recovery of protein func- tion. Importantly, this high-efficiency approach minimizes the number of reagents required to be introduced into patients’ cells and also circumvents homologous donor template delivery, which might be beneficial for transla- tion of HSPC gene editing to the clinic.
In the context of disease modeling, a more complex sce- nario would be to recreate the fusion proteins resulting from chromosomal rearrangements, a typical hallmark of some leukemias. CRISPR/Cas9-based editing has been successfully used in human cell lines and human HSC to generate chromosomal translocations resembling those described in acute leukemia, such as t(8;21)/RUNX1-ETO, t(4,11)/KMT2A-AFF1/AFF1-KMT2A and t(11;19)/MLL- ENL.30-32 This achievement is relevant because the model-
Table 4. Comparison of the different formats available for CRISPR/Cas9 components.
CRISPR format option
Cas9 and/or gRNA-encoding plasmids
Cas9 mRNA and gRNA
Ribonucleoprotein complex
Advantages Disadvantages
• Simple-to-use approach
• Multiple gRNA can be integrated into the same plasmid • Repositories available
• Economical
• Lack of genome integration
• Less off-targets than integrative plasmids
• Few off-target effects due to transient expression • Fast, avoiding cell transcription and/or translation • Highly efficient
• Off-targets from Cas9-constitutive expression
• Activation of innate immune system against plasmids
• Issues with mRNA stability • Immunogenicity
• Expensive
• Transient expression not sufficient in some contexts • More expensive than previous options
The main CRISPR component formats are: (i) DNA plasmids encoding the Cas9 protein and a guide RNA (gRNA), either individually or together; (ii) mRNA for Cas9 transla- tion applied to the cell,together with a separate gRNA.(iii) Ribonucleoprotein complexes,formed by pre-assembled Cas9 protein and gRNA.We highlight the most relevant pros and cons for each option. gRNA: guide RNA.
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haematologica | 2019; 104(5)