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Base editing for hemoglobinopathies testing double strand break free editing in
The current approaches for treating cultured cells.
hemoglobinopathies like Sickle Cell disease
(SCD) and Thalassemia rely on regular blood FnCas9 mediated editing in organoids
transfusions, iron chelation therapy and The current limitations of gene editing using
hydroxyurea that need to be administered CRISPR-Cas are due to the unintended off target
throughout life. The mortality rate due to effects and the DSBs (double strand breaks) that
hemoglobinopathies is still high especially in it introduces. Cas9 orthologue derived from
rural areas. Therefore, gene editing Francisella novicida (FnCas9), which has been
technologies are currently being explored for characterized in the lab, shows high mismatch
correcting the associated gene mutations. sensitivity and specificity. The application of
CRISPR-Cas based gene editing holds the FnCas9 in correcting gene mutation for SCD has
potential for therapeutic interventions, but the been demonstrated in an iPSC (induced
off-targeting effects and DSBs (double strand pluripotent stem cell) model as a proof of
breaks) limit its use in treatment at present. An concept for its translational applications. In the
alternative approach has been proposed in this present project, two conditions are being tested
project that utilizes engineered FnCas9 linked using FnCas9 that include generation of a
base editors (FnCas9-BE) to create a mutation murine ESC model of polyQ mutations and
that will increase the levels of fetal Hb in correction of the MLC mutation in an organoid
patients. FnCas9 was engineered by rational model of the disease. FnCas9 nickase (nFnCas9)
design to generate >50 combinations of was generated by site directed mutagenesis to
mutations in the PAM binding domains of the introduce D10A mutation along with inactive
enzyme (henceforth called enhanced FnCas9 or FnCas9 (dFnCAs9) by introducing two mutations
enFnCas9). Each of these combinations were in the HNH and RuvC domains (nuclease
validated by in vitro cleavage experiments to domains of Cas9 protein). Base editing
identify variants that have faster cleavage constructs, cytosine base editor (CBE) and
kinetics on a substrate as compared to the wild adenine base editor (ABE), were created by
type. Among the variants that performed fusing nFnCAs9 with CBE and ABE base editors.
optimally, enFnCas9-1, enFnCas9-15 and The transfection efficiency of these constructs
enFnCas9-31 all performed with up to 8-9 times was further compared by transfecting HEK-293T
more efficiency than the wild type variant and cells and found to be comparable to nSpCas9
were used for further studies. The motivation to base editors. Following sequence confirmation
develop more efficient FnCas9 variants arises of MLC patients recruited at AIIMS, iPSCs were
from the reduced accessibility to certain successfully generated from one of the MLC
genomic regions by wild type FnCas9. It was patient’s PBMCs. iPSCs were sorted using TRA1-
anticipated that by modifying the PAM 60 marker (using FACS) and were cryopreserved
interacting bases with more favorable for further experiments. For correcting the mlc
mutations, the binding affinity of the enFnCas9 gene mutation, FnCas9 based strategy was
variants to their targets would be improved developed. To achieve this, cloning of crRNA
leading to better editing outcomes. Expectedly, sequence targeting mlc1 gene mutation was
enFnCas9 variant 15 showed 3 times higher carried out and the sequence confirmed. Donor
binding affinity to the same substrate as template for correcting the mutation via HDR
compared to the wild type protein. Design of was next designed.
FnCas9 base editors has been done and
constructs have been generated and validated Cas9 delivery with nanoplexes
in cultured cells. The first generation WTFnCas9 Retinal dystrophy is the degeneration of retinal
base editors showed very low activity.; tissue due to heterogenous genetic mutations
enFnCas9 base editors are being constructed for that cause progressive loss of vision. Wet age-
Annual Report 2020-21 56
