Improving prime editing with an endogenous small RNA-binding protein

Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR–Cas guide RNAs. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase?III transcripts. We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein. Overall design: To study how La affects (e)pegRNAs, we transfected either targeting or nontargeting (e)pegRNA plasmids into K562 PEmax parental cells and La-knockout clonal cell line 4, constructed small RNA libraries from total RNA and performed paired-end sequencing. To study if PE7 affects gene expression as compared to PEmax or PE7 mutant, we transfected K562 cells with plasmids expressing PEmax, PE7 or PE7 mutant and pegRNA plasmid encoding HEK3 1TtoA or PRNP 6GtoT, constructed mRNA libraries from total RNA and performed single end sequencing. Related to GSE255003.

先导编辑（prime editing）可通过将模板序列逆转录至CRISPR–Cas向导RNA的3'端，实现基因组的精准修饰。为鉴定先导编辑的细胞决定因子，我们开发了可规模化的先导编辑报告系统，并开展了全基因组CRISPR干扰筛选。从这些筛选结果中，单一因子成为调控先导编辑效率最强的介导因子：小分子RNA结合外切核酸酶保护因子La。进一步研究显示，La可在多种编辑系统（PE2、PE3、PE4及PE5）、编辑类型（碱基替换、插入与缺失）、内源基因座及细胞类型中提升先导编辑效率，但对依赖标准未延伸向导RNA的基因组编辑方法无一致影响。既往研究表明，La可结合RNA聚合酶III转录本3'端的多聚尿苷酸序列。我们发现La可与多聚尿苷酸化的先导编辑向导RNA（prime editing guide RNA，pegRNAs）的3'端发生功能性相互作用。基于上述结果，我们开发了一种与La的RNA结合N端结构域融合的先导编辑蛋白（PE7）。该编辑器可提升表达型pegRNA、工程化pegRNA（engineered pegRNAs，epegRNAs）以及经La结合优化的合成型pegRNA的先导编辑效率。综上，我们的研究为先导编辑组分如何与细胞环境相互作用提供了关键见解，并提出了稳定细胞中外源小分子RNA的通用策略。

整体实验设计：为研究La对（工程化）先导编辑向导RNA的影响，我们将靶向或非靶向（工程化）先导编辑向导RNA质粒分别转染至K562 PEmax亲本细胞系以及La基因敲除单克隆细胞株4，从总RNA中构建小RNA文库并开展双端测序。为比较PE7与PEmax或PE7突变体对基因表达的影响，我们将表达PEmax、PE7或PE7突变体的质粒与编码HEK3 1T→A或PRNP 6G→T的先导编辑向导RNA质粒共转染至K562细胞，从总RNA中构建mRNA文库并开展单端测序。本数据集关联GSE255003。