give me one pargraph for analsys i and interptations
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need one pargraph for analsysis and interreataition this data hihgloihts the failure wher edited bcl11a resulted into hihger y globin rather than makeing it smaller than than untrettreated other than 65 Fig. 3. Consequences of targeted deletion on the protein level. Knockdown
of BCL11A expression leads to fetal hemoglobin induction. Western blot analysis was performed to determine HbF expression levels in BCL11A-edited K562
clones #65, #68, and #73 treated by the combination of sgRNAs A and C (A).
Quantification of western blots derived from 3 independent experiments using
ImageJ software (B). Data represent the mean ± S.D. of densitometric analysis
of immunoblots. g; guide RNA. The ZFN-mediated BCL11A knockout via disruption of the
GATAA motif in the erythroid enhancer does not impair on long-term
engraftment of HSCs as well as terminal erythroid maturation (Chang
et al., 2017). The accurate level of HbF for clinical utility is hypothesized to be ≥ 20% for SCD and slightly higher in β-thalassemia (Canver
and Orkin, 2016), moreover, reported that nearly 10%–20% of genetically modified HSCs are sufficient to have clinical improvement in
thalassemia intermediate and thalassemia major, respectively
(Antoniani et al., 2018; Brendel et al., 2016; Chang et al., 2017;
Persons, 2001). Studies using single gRNAs were shown to induce deletions up to 600 bp and 1.5 kb in mouse zygotes and haploid cancer
cell lines, respectively. Moreover, NHEJ-based indels are usually not
enough to loss of function of non-coding genes, gene clusters or regulatory sequences. However, inversion events have not been reported
using single gRNA. Whereas, genomic deletion with two sgRNAs is
more favorable for loss of function of non-coding genes, gene clusters or
regulatory sequences. But there are some notable points in using pair
sgRNAs, larger-than-expected deletions (Canver et al., 2014; Kosicki
et al., 2018; Xiao et al., 2013) and inversion of large sequences of DNA
(Shin et al., 2017). The deleted of small DNA segments have not an
opportunity to be re-integrated into the genome like the large segments,
presumably because they immediately degraded by exonucleases. In
our experiment, we assumed that the deletion of the 0.2 kb of intronic
DNA region could exclude the inversion event in comparison with
1.5 kb or more than that (Canver et al., 2015). Inthe present study, we
investigated a potential strategy to reproduce the expression of fetal
hemoglobin via targeted deletion of the GATAA motif and flanking
sequences of that which is including the functional core sequence of the
+58 DNaseI hypersensitive site (DHS) within the BCL11A erythroid
enhancer. In this regard, we designed two sgRNAs based on SaCas9 and
four sgRNA based on SpCas9 (Bauer et al., 2013; Canver et al., 2015;
Vierstra et al., 2015). Identical deletions had no effect of HbF induction
in the bulk population and edited clones of KG-1 or KU812 cell lines
even when carrying homozygous deletions, suggesting that these cells
presumably not a potential model for screening HbF reactivation
especially via BCL11A knockout approaches (Zein et al., 2010), or due
to they are cancer-derived cell lines with abnormal genome and DNA
repair mechanisms (Kosicki et al., 2018). S. aureus Cas9-mediated
genome editing has advantages for efficient delivery because it is
around 1 Kb smaller than SpCas9, and it recognizes a longer protospacer-adjacent motif (PAM) 5′-NNG (A/G) (A/G) T-3′. Thus, to improve On-target effect we chose SaCas9 in addition to SpCas9 for
genome editing (Friedland et al., 2015; Ran et al., 2015; Ye et al.,
2016). While initial experiments with VVT1 did not drive any indel
mutations (Benjamin P Kleinstiver et al., 2015a,b), we altered tracrRNA
and made a new version with smaller tracrRNA sequence, 81bp, entitled modified VVT1 (mVVT1) leads to 9% deletion frequency in
treated HEK293T cells with a combination of gαI and gαII. We used the
same constructs into the erythroid cell lines and unexpectedly realized
no indel mutations, suggesting that probably the SaCas9-mediated
editing obstructed by the chromatin context (Daer et al., 2017;
Kallimasioti-Pazi et al., n.d.; Yarrington et al., 2018). The frequency of
the HDR-mediated genome editing strategies to correct the SCD mutations in HSPCs was up to 40% suggesting that HDR might be less acceptable in HSCs. Thus, the clinical utilization of genome editing could
be hindered for diseases requiring a high frequency of modification and
correction. Contrary, the NHEJ-based approaches might be more successful because this strategy is donor template independent and likely
more common than HDR in these cells (Dewitt et al., 2016; Genovese
et al., 2014; Gundry et al., 2016; Hoban et al., 2015; Megan D Hoban
et al., 2016a,b). Our results indicated that targeted disruption of a tiny
region as small as a 200bp region of BCL11A gene leads to a significant
induction of γ -globin expression and reproduction of fetal hemoglobin
in K562 a human erythroleukemia cell line. In our experiment, we
realized that the combination of pMAK-gA + gC has more potential in
HbF reactivation as well as on-target deletion. The precise predicted
deletion occurred in 66.6% (4/6) of biallelic deleted clones. In addition,
fetal hemoglobin reactivation significantly induced in 18.75% (3/16)
and 10% (1/10) of genetically edited clones using transfection of
gA + gC (clones #63, #65, and #73), and gA + gD (clone #82) respectively (Fig. 3). Western blotting data for clones #63 and #82 are
not shown.
5. Conclusion
This study demonstrates that even a small CRISPR-Cas9-based
genomic deletion within the BCL11A enhancer in the second intron
could be able to activate the adult to fetal hemoglobin reverse switching
and fetal hemoglobin synthesis. Ultimately this approach may be considered as a potentially curative gene therapy approach for βhemoglobinopathies.Acknowledgments
This work was supported by grants from Pasteur Institute of Iran
[grant number: BP-9035] and was done in Museum National d’Histoire
Naturelle, INSERM U1154, CNRS UMR 7196, Sorbonne Universités, 43
rue Cuvier, Paris F-75231, France and Institute of Laboratory Animal
Science, University of Zurich, Switzerland. The authors would like to
thanks Jean-Baptiste-Renaud in Dr. Jean-Paul Concordet lab and
Stanislav Pantelyushin in Prof. Dr. Thorsten Buch lab for their collaborations.
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The data presented in the study highlights the unexpected outcome of editing the BCL11A gene, resulting in higher levels of γ-globin expression rather than the anticipated reduction. This unexpected result underscores the complexity of gene editing techniques and the unpredictability of genetic manipulation. The study also emphasizes the importance of carefully designing and executing gene editing experiments, as even small deletions within enhancer regions can have significant impacts on gene expression and protein levels. Overall, these findings suggest that targeted genomic deletions can be a promising strategy for inducing fetal hemoglobin synthesis, offering potential therapeutic implications for β-hemoglobinopathies. Further research is needed to fully understand the mechanisms underlying these outcomes and to optimize gene editing strategies for clinical applications.
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