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CN106011171A 一种利用CRISPR/Cas9技术基于SSA修复的基因无缝编辑方法 失效 - 权利终止

一种利用CRISPR/Cas9技术基于SSA修复的基因无缝编辑方法
Abstract:
本发明主要属于基因编辑技术领域，具体涉及一种利用CRISPR/Cas9技术基于SSA修复的基因无缝编辑方法。所述基因无缝编辑方法首先构建靶向CCR5和eGFP的CRISPR/Cas9表达载体CCR5. CRISPR/Cas9和eGFP.CRISPR/Cas9，并构建供体CCR5‑Donor载体，所述CCR5‑Donor质粒载体具有pXL‑BACII‑L‑arm‑CAG‑TK‑PGK‑PuroR‑T2A‑eGFP‑bGH pA ‑R‑arm结构，其中，L‑arm和R‑arm为左右同源臂，L‑arm和R‑arm中间的CAG‑TK‑PGK‑PuroR‑T2A‑eGFP‑bGH pA为筛选标记组件；通过两次转染，利用CRISPR‑Cas9系统及细胞内的HR和SSA修复机制，通过细胞的嘌呤霉素和更昔洛韦正负向筛选，完成无缝编辑。
Abstract(English):

The invention mainly belongs to the technical field of gene editing and particularly relates to an SSA (single-strand annealing) repair-based gene seamless editing method utilizing a CRISPR/Cas9 technology. According to the gene seamless editing method, CRISPR/Cas9 expression vectors CCR5.CRISPR/Cas9 and eGFP.CRISPR/Cas9 targeting CCR5 and eGFP are constructed firstly, a donor CCR5-Donor vector is constructed, a CCR5-Donor plasmid vector adopts the pXL-BACII-L-arm-CAG-TK-PGK-Puro-T2A-eGFP-bGH pA-R-arm structure, L-arm and R-arm are left and right homologous arms, and CAG-TK-PGK-Puro-T2A-eGFP-bGH pA between L-arm and R-arm is a screening marking component; through two times of transfection, seamless editing is completed by using HR and SSA repair mechanisms in the CRISPR-Cas9 system and cells through positive and negative screening of puromycin and ganciclovir of the cells.

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