Bamidele Nathan, Ansodaria Aditya, Chen Zexiang, Cheng Haoyang, Panwala Rebecca, Jazbec Eva, Sontheimer Erik J
RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, Massachusetts, 01605, USA.
Current address: Profluent, Emeryville, CA 94608, USA.
bioRxiv. 2024 Nov 7:2024.10.30.620986. doi: 10.1101/2024.10.30.620986.
CRISPR-Cas genome editing tools enable precise, RNA-guided modification of genomes within living cells. The most clinically advanced genome editors are Cas9 nucleases, but many nuclease technologies provide only limited control over genome editing outcomes. Adenine base editors (ABEs) and cytosine base editors (CBEs) enable precise and efficient nucleotide conversions of A:T-to-G:C and C:G-to-T:A base pairs, respectively. Therapeutic use of base editors (BEs) provides an avenue to correct approximately 30% of human pathogenic variants. Nonetheless, factors such as protospacer adjacent motif (PAM) availability, accuracy, product purity, and delivery limit the full therapeutic potential of BEs. We previously developed Nme2Cas9 and its BE derivatives, including ABEs compatible with single adeno-associated virus (AAV) vector delivery, in part to enable editing near NCC PAMs. Further engineering yielded domain-inlaid BEs with enhanced activity, as well as Nme2Cas9/SmuCas9 chimeras that target single-cytidine (NC) PAMs. Here we further enhance Nme2Cas9 and Nme2Cas9 editing effectors for improved efficiency and vector compatibility through site-directed mutagenesis and deaminase linker optimization. Finally, we define the editing and specificity profiles of the resulting variants by using paired guide-target libraries.
CRISPR-Cas基因组编辑工具能够在活细胞内对基因组进行精确的、RNA引导的修饰。临床上最先进的基因组编辑器是Cas9核酸酶，但许多核酸酶技术对基因组编辑结果的控制有限。腺嘌呤碱基编辑器（ABE）和胞嘧啶碱基编辑器（CBE）分别能够将A:T碱基对精确高效地转换为G:C碱基对，以及将C:G碱基对精确高效地转换为T:A碱基对。碱基编辑器（BE）的治疗应用为纠正约30%的人类致病变体提供了一条途径。尽管如此，诸如原间隔相邻基序（PAM）可用性、准确性、产物纯度和递送等因素限制了BE的全部治疗潜力。我们之前开发了Nme2Cas9及其BE衍生物，包括与单腺相关病毒（AAV）载体递送兼容的ABE，部分原因是为了实现靠近NCC PAM的编辑。进一步的工程改造产生了具有增强活性的结构域镶嵌BE，以及靶向单胞嘧啶（NC）PAM的Nme2Cas9/SmuCas9嵌合体。在这里，我们通过定点诱变和脱氨酶接头优化进一步增强Nme2Cas9和Nme2Cas9编辑效应器，以提高效率和载体兼容性。最后，我们通过使用配对的向导-靶标文库来定义所得变体的编辑和特异性图谱。
