Detection of CRISPR/Cas9-Mediated Fetal Hemoglobin Reactivation in Erythroblasts Derived from Cord Blood-Hematopoietic Stem Cells.

Reactivation of the fetal hemoglobin (HbF) in adult erythroid cells via genome editing is a strategy for the treatment of ß-thalassemia and sickle cell disease. In related reports, the reactivation of HbF is regularly examined in erythroblasts which are generated from the adult CD34+ hematopoietic stem and progenitor cells (HSPCs). However, the procurement of adult HSPCs, either from the bone-marrow (BM) or from mobilized peripheral-blood (mPB), is difficult. Cord-blood (CB) is a readily available source of HSPCs. CB-HSPCs, however, produce high quantities of HbF following differentiation into the erythroid lineage-a potential drawback in such studies. Here, we have edited the BCL11A enhancer (a well-characterized HbF-quantitative trait loci or QTL) via CRISPR/Cas9 in order to determine whether HbF reactivation could be detected in CB-HSPC-derived erythroblasts. In the edited erythroblasts, insertion/deletion (indel) frequencies of 74.0-80.4% and BCL11A RNA reduction levels of 92.6 ± 5.1% (P < 0.0001) were obtained. In turn, the γ/ß-globin transcript ratios were increased from 11.3 ± 1.1-fold to 77.1 ± 2.0-fold, i.e., by 6.8-fold (P < 0.0001)-and the HbF% levels increased from 34.3% in the control population to 43.5% in the BCL11A edited erythroblasts. Our results suggest that γ-globin/HbF reactivation via genome editing can be detected in CB-HSPCs generated erythroblasts-rendering CB-HSPCs a useful model for similar studies.

Pitfalls of Restriction Enzyme Mapping Following Generation of CRISPR Constructs.

BACKGROUND: The PX330 and the related PX459 plasmids are widely used for Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-mediated genome editing. Screening for plasmids containing the correct sgRNA template insertion is one of the most important steps in this system. Different methods for screening the sgRNA inserts have been deployed. One such method is Restriction Enzyme (RE) mapping. Restriction enzyme mapping can be used to screen for numerous plasmid recombinants simultaneously. METHODS: In this study, the sgRNA templates were initially cloned into the above PX459 plasmids. Subsequently, the accuracy of the constructs was determined by RE mapping. RESULTS: This method was established to screen for sgRNA-bearing PX459 plasmids. However, numerous anomalies were detected after ligation of sgRNA templates into RE digested PX459 plasmids. CONCLUSION: Our data suggest that RE mapping is only appropriate as an initial screen and that the identity of all plasmids with the correctly identified RE maps should be confirmed by Sanger sequencing.

An enhancer haplotype may influence BCL11A expression levels and the response to hydroxyurea in ß-thalassemia patients.

AIM: To identify the BCL11A intron-2 enhancer linkage disequilibrium (LD) block, harboring two previously identified SNPs, associating with the hydroxyurea response in ß-thalassemia patients and the functional significance of this region. MATERIALS & METHODS: Several neighboring SNPs were genotyped in our cohort. The associating LD block was identified, and its function studied in K562 erythroid cells via CRISPR/Cas9 genome editing. RESULTS: A haplotype harboring three tag SNPs correlated significantly with the HU-response and BCL11A transcript levels in the patients' reticulocytes. Two deletions encompassing this LD block significantly reduced BCL11A transcript levels in K562 cells. CONCLUSION: Our data suggest an essential role for this LD block in BCL11A expression levels and the response to hydroxyurea in ß-thalassemia patients.