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.

CAS9和CAS12A等CRISPR效应已成为生物医学研究的强大工具 为了引入活细胞中的靶向突变，因此，对于这种能力，它们具有明显的治疗性 治疗遗传疾病的潜力 - 尽管存在明显的临床风险，但他们可能会引入“ 目标’或意外突变。而CRISPR效应的力量在于，它们的序列是 公认和目标是其RNA辅助因子的模块化的“可编程”段（它们的 “引导RNA”或GRNA），它们的突变活性可以在不完美的核苷酸序列上触发 不可预测，对他们的grnas的互补性也是如此。显然，不受控制的突变的可能性会增加 患者和临床医生的危险信号，到目前为止，CRISPR基因疗法一直集中在高度上 专门的遗传情况。需要进一步改进CRISPR特异性，不仅要减轻 临床风险，但也要推动CRISPR的新应用 - 例如，如果单核苷酸变体（SNV） 可以可靠地歧视，它将允许对常染色体显性疾病的特定等位基因特异性基因编辑， 我们经常需要区分“健康”和 “疾病”等位基因，但是当前的CRISPR技术无法始终如一。我们最近证明了 能够通过刻板命令提高CRISPR效应子特异性的方法的可行性，并且 以某种方式可以协同应用于许多其他以前开发的技术 进一步提高特异性。通过向GRNA（X-GRNA）添加额外的核苷酸并设计扩展 用GRNA的DNA靶向段形成“发夹”二级结构的顺序 HP-grnas）使与脱离目标的互动不稳定，我们可以生成X/HP-GRNA，从而显着限制 脱离目标活动，同时保持crispr效应变体中的靶向突变活动，从四个 不同的生物和一种工程衍生物。因此，长期目标是了解 在X-GRNA中设计扩展序列，这将导致超特定性的CRISPR效应 在任何可CRISPR的网站上。为了实现这一目标，在此R21中，我们将执行一个详尽的屏幕 靶向不同临床相关位点的随机X-GRNA文库，并确定什么共同序列 这些X-GRNA的二级结构特征和/或二级结构特征驱动了特异性的显着增加。而 该建议的风险是，对于所有X/HP-Grna设计，可能没有“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，以便研究人员经验 为任何CRISPR效应子生成超特异性X-GRNA。可能的回报是 X-GRNA（具有工程CRISPR效应）的协同使用有效地消除了风险 在CRISPR应用中意外突变，以及将我们的高通量方法与机器相结合的 学习将允许任何人生产新的计算工具。