Page 49 - Annual report 2021-22

Annual Report 2021-22 |

Debojyoti Chakraborty

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Tackling Sickle Cell Anemia, a genetic disease prevalent in many tribal populations in central India is a
national mission. At IGIB, Debojyoti Chakraborty has taken up development of gene editing
technologies, specially those focussed on engineering Cas9 proteins to improve fundamental
knowledge of genetic correction as well as tools for design and implementation in clinical trials. FnCas9
was engineered by rational design to generate >50 combination of mutations in the PAM binding
domains of the enzyme (henceforth called enhanced FnCas9 or enFnCas9), in collaboration with the
lab of Prof. Osamu Nureki at University of Tokyo, Japan. A specific goal is the development of enFnCas9
suitable for ex vivo gene editing and validation at Sickle Cell locus. The variants of enFnCas9 harboring
novel mutations that increase PAM interactions without compromising on specificity have been
constructed. These variants show up to 60% editing in mammalian cells studied in the lab, which is
well over the minimum clinical requirements. The enFnCas9 proteins also show higher editing than
naturally occurring FnCas9 or any of the high fidelity SpCas9 proteins (SpCas9-HF1 or eSpCas9) that
have been previously engineered for greater specificity. None of the well characterized off-targets
show any editing activity suggesting the high specificity of these variants and suitability for preclinical
testing. Currently enFnCas9 Base editors are being constructed for cellular measurements of double
strand break free editing.

The enFnCas9 variants have been tested for Homology Directed Repair (HDR) at multiple loci and show
a higher HDR rate of genetic incorporation than FnCas9 or SpCas9-HF1/eSpCas9. It has been tested
for sickle cell locus correction in mammalian cells. Importantly, the HDR/NHEJ rate of FnCas9 is higher
than SpCas9 showing that non-specific indels (which can lead to beta thalassemia) is lower when
FnCas9 is used. This provides a safer therapeutic potential for targeted base correction.

FELUDA, a CRISPR based diagnostic system first deployed for detection of SARS CoV2 can also utilize
the enhanced features of enFnCas9. On lateral flow assay, enFnCas9 variants exhibit higher binding
affinity to cognate substrates as seen through microscale thermophoresis experiments. Owing to this
it exhibited stronger signal on a lateral flow assay and was able to achieve a higher resolution of single
nucleotide polymorphism detection using FELUDA/RAY. Importantly, the more flexible PAM
recognition coupled with greater signal strength has now made it possible to use FELUDA for SNV
diagnosis beyond the standard NGG PAM containing regions (>85% of all Mendelian SNVs).

A unified webserver CriSNPr (CRISPR based SNP recognition), which provides the user the opportunity
to de-novo design gRNAs based on six CRISPRDx proteins of choice (Fn/enFnCas9, LwCas13a,
LbCas12a, AaCas12b, and Cas14a) and query for ready-to-use oligonucleotide sequences for validation
on relevant samples was developed. In addition, a database of curated pre-designed gRNAs and
target/off-target for all human and SARS-CoV-2 variants reported so far has also been provided.
CriSNPr has been validated on multiple Cas proteins and highlights its broad and immediate scope of
utilization across multiple detection platforms. CriSNPr is available at URL http://crisnpr.igib.res.in/.

Besides the central goal of developing Cas9 variants with improved properties, Debojyoti Chakraborty
has been supporting various studies in collaboration with other researchers in IGIB and across India
to address challenges in taking such a high-end technology to the market. This includes the