Department of Medicine, Vascular Biology Center, Medical College of Georgia at Augusta University, Augusta, GA, 30912, USA.
Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, 38195, USA.
Genome Biol. 2021 Mar 16;22(1):83. doi: 10.1186/s13059-021-02304-3.
Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the Tspan2 promoter.
Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders.
PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.
大多数单核苷酸变异（SNV）发生在非编码序列中，那里有数百万个转录因子结合位点（TFBS）。在这里，对 CRISPR 介导的同源定向修复（HDR）与最近报道的 Prime Editing 2（PE2）系统在小鼠中的一个称为 Tspan2 启动子中的 CArG 盒的 TFBS 上进行了比较分析。
定量 RT-PCR 显示，在 Tspan2 CArG 盒中发生 HDR 介导的三个碱基替换的纯合小鼠的主动脉和膀胱中 Tspan2 mRNA 丢失，但心脏和大脑中没有丢失。使用相同的原间隔区，在 Tspan2 CArG 盒中发生 PE2 介导的单碱基替换的纯合小鼠中也显示出类似的细胞特异性 Tspan2 mRNA 丢失；重叠的长非编码 RNA 的表达也几乎在主动脉和膀胱中被废除。免疫 RNA 荧光原位杂交验证了 HDR 和 PE2 CArG 盒突变小鼠中 Tspan2 在血管平滑肌细胞中的丢失。靶向测序显示所有 PE2 和 HDR 创始者中都存在可变频率的靶标编辑。然而，尽管在任何 PE2 创始者中都未检测到靶标插入缺失，但所有 HDR 创始者均显示出靶标插入缺失的不同水平。通过靶向测序进行的脱靶分析显示，许多 HDR 创始者发生了突变，但在 PE2 创始者中没有发现突变。
PE2 指导 TFBS 中单碱基的高精度编辑，导致靶标 mRNA/长非编码 RNA 基因对的细胞特异性表达丢失。PE2 平台扩展了基因组编辑工具包，用于在小鼠中模拟和纠正相关的非编码 SNV。
