Page 60 - CSIR-IGIB Annual Report 2020-21

related macular degeneration (wAMD) and currently available CRISPR-Cas gene editing.
Leber congenital amaurosis (LCA) are two such VEGF A and RPE65 gene for wAMD and LCA
retinal diseases due to mutations in VEGFA and disease are being targeted, respectively.
RPE65 genes respectively. The current use of Therefore, sgRNA were designed with respect to
gene editing technologies like CRISPR-Cas are the target gene using available software. In vitro
limited by the delivery of the enzyme complexes transcription based sgRNA synthesis was
into the target cells besides off targeting and preferred, thus forward primer and universal
DSBs. This project utilizes lipopolymeric reverse primer were designed to target the
nanoplexes of CRISPR/Cas9 ribonucleoprotein VEGFA and RPE65. Herein, more than two
(RNP) complex to correct these disease-causing sgRNA were designed for one target gene in
mutations. FnCas9 which has a high mismatch order to evaluate the specificity. Both, eGFP
sensitivity and specificity with very less off- tagged as well as normal Cas9 protein were
target effects is being deployed in this approach purified and characterized for its endonuclease
to overcome the other two limitations of activity.

Publications
Role of lncRNAs in stem cell maintenance and differentiation. Aich M, Chakraborty D. Curr Top Dev Biol.
2020;138:73-112.

Terminal uridylyl transferase mediated site-directed access to clickable chromatin employing CRISPR-dCas9.
George JT, Azhar M, Aich M, Sinha D, Ambi UB, Maiti S, Chakraborty D, Srivatsan SG. J Am Chem Soc. 2020 Aug
12;142(32):13954-13965.

Ophthalmic genetics practice and research in India: Vision in 2020. Bansal M, Tandon R, Saxena R, Sharma A, Sen
S, Kishore A, Venkatesh P, Maiti S, Chakraborty D. Am J Med Genet C Semin Med Genet. 2020 Sep;184(3):718-727.

Voices of biotech research. Annabi N, Baker M, Boettiger A, Chakraborty D, Chen Y, Corbett KS, Correia B, Dahlman
J, de Oliveira T, Ertuerk A, Yanik MF, Henaff E, Huch M, Iliev ID, Jacobs T, Junca H, Keung A, Kolodkin-Gal I,
Krishnaswamy S, Lancaster M, Macosko E, Martínez-Núñez MA, Miura K, Molloy J, Cruz AO, Platt RJ, Posey AD Jr,
Shao H, Simunovic M, Slavov N, Takebe T, Vandenberghe LH, Varshney RK, Wang J. Nat Biotechnol. 2021
Mar;39(3):281-286.

Preprint
Rapid, accurate, nucleobase detection using FnCas9 Mohd. Azhar, Rhythm Phutela, Manoj Kumar, Asgar Hussain
Ansari, Riya Rauthan, Sneha Gulati, Namrata Sharma, Dipanjali Sinha, Saumya Sharma, Sunaina Singh, Sundaram
Acharya, Deepanjan Paul, Poorti Kathpalia, Meghali Aich, Paras Sehgal, Gyan Ranjan, Rahul C. Bhoyar, Indian CoV2
Genomics & Genetic Epidemiology (IndiCovGEN) Consortium, Khushboo Singhal, Harsha Lad, Pradeep Kumar Patra,
Govind Makharia, Giriraj Ratan Chandak, Bala Pesala, Debojyoti Chakraborty, Souvik Maiti medRxiv
2020.09.13.20193581

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