A Molecular Grammar for Guide RNAs (gRNAs) with Engineered Secondary Structures

具有工程化二级结构的向导 RNA (gRNA) 的分子语法

• 批准号: 10683334
• 负责人: Eric Alan Josephs
• 金额: $ 21.83万
• 依托单位: UNIVERSITY OF NORTH CAROLINA GREENSBORO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10683334
• 关键词: Affect; Alleles; Base Sequence; Biomedical Research; Biophysics; Biotechnology; CRISPR/Cas technology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Computer Analysis; DNA; Data Set; Development; Directed Molecular Evolution; Disease; Dominant Genetic Conditions; Engineering; Event; Familial Hypercholesterolemia; Genes; Genetic; Genetic Diseases; Goals; Guide RNA; Hand; Hereditary Spherocytosis; Heterozygote; Human; Intuition; Machine Learning; Molecular; Mutation; Nature; Nucleic Acids; Nucleotides; Oncogenes; Organism; Patients; Proteins; Protocols documentation; RNA; RNA Sequences; RNA library; Randomized; Research; Research Personnel; Rewards; Risk; Series; Single Nucleotide Polymorphism; Site; Specificity; Structure; Techniques; Therapeutic; Training; Variant; Work; autosome; clinical risk; clinically relevant; cofactor; computerized tools; design; gene therapy; genetic variant; genome-wide; high throughput screening; improved; interest; machine learning model; member; microorganism; novel; novel therapeutics; off-target mutation; off-target site; prevent; therapeutic target; tool

CRISPR effectors like Cas9 and Cas12a have emerged as powerful tools in biomedical research for their ability to introduce targeted mutations in living cells and, consequentially, for this ability they hold significant therapeutic potential for treating genetic disorders—despite also carrying significant clinical risk that they may introduce ‘off- target’ or unintended mutations. While the power of CRISPR effectors lies in the fact that the sequences they recognize and target are complementary to a modular, ‘programmable’ segment of their RNA cofactors (their ‘guide RNAs’ or gRNAs), their mutational activity can be triggered at nucleotide sequences with imperfect complementarity to their gRNAs as well, unpredictably. Obviously, the possibility of uncontrolled mutation raises red flags for both patients and clinicians and so far, CRISPR gene therapies have been focused on highly specialized genetic situations. Further improvements to CRISPR specificity are necessary, not only to mitigate clinical risk, but also to drive new applications of CRISPR—for example, if single nucleotide variants (SNVs) could be reliably discriminated, it would allow for allele-specific gene editing of autosomal dominant disorders, where often we would need to discriminate between small sequence variations between the ‘healthy’ and ‘disease’ alleles but which current CRISPR technologies cannot consistently do. We recently demonstrated the feasibility of an approach that is capable of improving CRISPR effector specificity by orders-of-magnitude, and in such a way that it can be synergistically applied to many of the other previously-developed techniques to improve specificity further. By adding extra nucleotides to the gRNA (x-gRNA) and designing the extended sequence to form ‘hairpin’ secondary structures with the DNA-targeting segment of the gRNA (hairpin-gRNAs or hp-gRNAs) that destabilize interactions with off-targets, we could generate x/hp-gRNAs that significantly limited off-target activity while maintaining on-target mutational activity in CRISPR effector variants derived from four different organisms and one engineered derivative. The long-term goal is therefore to understand the rules for designing extended sequences in x-gRNAs that would result in ultra-specificity for divergent CRISPR effectors at any CRISPR-targetable site. To achieve that goal, in this R21 we will perform an exhaustive screen of randomized x-gRNA libraries targeting different clinically-relevant sites and identify what common sequence and/or secondary-structure features of those x-gRNAs drive significant increases in specificity. While the riskiness of this proposal is that there may not be “universal design rules”, per se, for all x/hp-gRNA designs and targets, this work will nevertheless provide a practical (design-free) platform for researchers to empirically generate ultra-specific x-gRNAs for any target of interest for any CRISPR effector. The likely reward is that synergistic use of x-gRNAs (with engineered CRISPR effectors) has the potential to effectively abrogate the risk of unintended mutation in CRISPR applications, and that combining our high-throughput approach with machine- learning would allow new computational tools for anyone to produce, de novo, ultra- or allele-specific x-gRNAs.

CRISPR效应物如Cas9和Cas 12 a已经成为生物医学研究中的强大工具，因为它们能够 在活细胞中引入靶向突变，因此，对于这种能力，它们具有重要的治疗作用。 治疗遗传性疾病的潜力-尽管也具有重大的临床风险，它们可能会引入“关闭”， 靶向或非预期突变。虽然CRISPR效应子的力量在于它们所表达的序列 识别和靶向与它们的RNA辅因子的模块化的、“可编程的”片段互补（它们的 在一些实施方案中，当突变体（例如，“指导RNA”或gRNA）发生突变时，它们的突变活性可以在具有不完全互补性的核苷酸序列处被触发。 与其gRNA的互补性也是不可预测的。很明显，不受控制的突变的可能性 对患者和临床医生来说都是危险信号，到目前为止，CRISPR基因疗法已经被高度关注， 特殊的基因情况。CRISPR特异性的进一步改进是必要的，不仅是为了减轻 临床风险，但也推动CRISPR的新应用-例如，如果单核苷酸变异（SNV） 可以可靠地区分，它将允许常染色体显性遗传病的等位基因特异性基因编辑， 我们经常需要区分“健康”和“健康”之间的小序列变异， “疾病”等位基因，但目前的CRISPR技术无法始终如一地做到这一点。我们最近展示了 能够以数量级改善CRISPR效应子特异性的方法的可行性，以及 以这种方式，它可以协同地应用于许多其他先前开发的技术， 进一步提高了特异性。通过向gRNA（x-gRNA）添加额外的核苷酸并设计延伸的 序列以与gRNA的DNA靶向区段形成“发夹”二级结构（发夹-gRNA或 hp-gRNA），使与脱靶的相互作用不稳定，我们可以产生显著限制 CRISPR效应子变体中的脱靶活性，同时维持中靶突变活性， 不同的生物体和一种工程衍生物。因此，长期目标是了解 设计x-gRNA中的延伸序列，从而对不同的CRISPR效应子产生超特异性 在任何CRISPR靶向位点。为了实现这一目标，在本R21中，我们将对 随机化的x-gRNA文库靶向不同的临床相关位点，并确定哪些共同序列 和/或二级结构特征驱动特异性的显著增加。而 这一提议的风险在于，对于所有x/hp-gRNA设计本身，可能不存在“通用设计规则” 和目标，这项工作将提供一个实用的（无设计）平台，研究人员经验 产生针对任何CRISPR效应子的任何目标靶标的超特异性x-gRNA。可能的回报是， 协同使用x-gRNA（与工程化CRISPR效应子）有可能有效地消除 CRISPR应用中的意外突变，以及将我们的高通量方法与机器结合起来， 学习将允许任何人使用新的计算工具来从头产生超或等位基因特异性x-gRNA。