ĐÁNH GIÁ HOẠT TÍNH CHỈNH SỬA GEN BCL11A TRÊN THỰC NGHIỆM CỦA PROTEIN CAS9 TÁI TỔ HỢP, ĐỊNH HƯỚNG ỨNG DỤNG ĐIỀU TRỊ BỆNH HỒNG CẦU LIỀM

Abstract

Objective: To design a rCas9/sgRNA complex for editting of BCL11A gene in in-vitro and evaluation of recombinant Cas9 protein activity for orientational applying in the treatment of sickle cell disease. Materials and methods: Amplification of the enhancer region of the BCL11A gene by PCR, sequencingand comparing with Vietnamese DNA. Design a single-strand guide RNA (sgRNA) and create an rCas9/sgRNA complex. Evaluate the activity rCas9/sgRNA complex on the BCL11A gene cloned into the pJET1.2 plasmid under in vitro condition. Results: The synthesized rCas9/sgRNA complex has cleaved the enhancer region of BCL11A gene in in-vitro into two products with the size of approximately 240bp. The gene sequencing showed that the rCas9/sgRNA complex cut exactly the GATAA enhancer region of BCL11A gene at a position of 3 nucleotides away from the PAM region according to theoretical calculations. Conclusion: Recombinant Cas9 protein had a similar activity as natural Cas9 protein and rCas9/sgRNA complex needs to be further studied for application in BCL11A gene editing to treat sickle cell disease in clinical practice.