Decorating chromatin for precise genome editing using CRISPR Cas

使用 CRISPR Cas 修饰染色质以进行精确的基因组编辑

• 批准号: 10230885
• 负责人: Enrique Lin Shiao
• 金额: $ 2.04万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2021-09-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10230885
• 关键词: Address; Affect; Architecture; Benchmarking; Biology; CRISPR/Cas technology; Cancer Model; Cell Line; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Double Strand Break; DNA Insertion Elements; DNA Repair; DNA Repair Pathway; Dependence; Deposition; Disease model; Engineering; Environment; Enzymes; Family; Family member; Flow Cytometry; Generations; Genes; Genetic; Genetic Diseases; Genetic Recombination; Genome; Genomics; Goals; Guide RNA; Histones; Hot Spot; Human; Immunotherapy; Knock-out; Mammalian Cell; Mediating; Meiosis; Methyltransferase; Nonhomologous DNA End Joining; Nucleosomes; Occupations; Organism; Outcome; Pathology; Pattern; Plants; Positioning Attribute; Process; Property; Proteins; Publications; Research Project Grants; Research Proposals; Ribonucleases; Role; Site; System; T cell therapy; T-Cell Receptor; T-Lymphocyte; Technical Expertise; Techniques; Testing; Training; Transgenes; Work; Yeasts; base; cancer cell; cell type; chimeric antigen receptor; chromatin modification; engineered T cells; experimental study; genome editing; genome-wide; histone modification; improved; insertion/deletion mutation; insight; next generation; next generation sequencing; novel; novel strategies; nuclease; precise genome editing; repaired; tool; tool development; transcriptome sequencing

Project Summary / Abstract CRISPR associated (Cas) systems have revolutionized biology by allowing the introduction of targeted double stranded DNA breaks (DSB). However, in human cells, non-homologous end joining (NHEJ) is the preferred DSB repair process which often leads to small insertions and deletions (indels) at the site of CRISPR-Cas- induced DSBs. As a result, CRISPR systems are efficient tools for creating genetic knockouts but making targeted insertions remains a challenge. Efficient targeted insertions would allow for quick generation of cancer models and would also pave the way for next generation adoptive T cell therapies, where chimeric antigen receptor (CAR) transgenes could be inserted at specific genomic sites that improve their ability to destroy cancer cells. Homology driven repair (HDR) mediated by donor templates is currently the best way to introduce precise edits and DNA insertions following CRISPR-Cas cutting, but the efficiency is generally low relative to NHEJ, and strategies to improve it have yielded only small advancements. Here, I propose an approach to improve HDR by making chromatin more accessible at the CRISPR-Cas induced cut site and decorating adjacent nucleosomes with H3K36me3, a histone mark known to be involved in mediating HDR. To this end, I have engineered a Cas9- PRDM9 fusion construct. PRDM9 is a methyltransferase that deposits H3K4me3 and H3K36me3 to mark recombination hot spots during meiosis and has also been shown to act as a pioneer factor making chromatin more accessible for downstream processes. In preliminary experiments, this novel fusion construct displays a 2-fold improvement in HDR:indel ratio compared to Cas9 alone. By directly altering chromatin architecture, I hypothesize that HDR levels can be increased for precise genome editing and targeted DNA insertions across different cell types and regardless of preexisting chromatin architecture. In the first aim, I plan to investigate how site-specific chromatin modifications mediate DNA repair following cutting by CRISPR-Cas9. I will also study whether direct reconfiguration of chromatin architecture via Cas9-PRDM9 fusion can improve homologous directed repair (HDR). In the second aim, I will investigate the effect on HDR of blunt vs staggered cuts introduced by different Cas12 family nucleases, including a newly identified hypercompact CasΦ, and whether fusions of these editors with PRDM9 can improve precise genome editing. Finally, I will investigate how these combined properties can be harnessed to improve large DNA insertions (>100bp) in both cell lines and human primary T cells.

项目总结/摘要 CRISPR相关（Cas）系统通过允许引入靶向双链RNA，彻底改变了生物学。 DNA链断裂（DSB）。然而，在人细胞中，非同源末端连接（NHEJ）是优选的。 DSB修复过程通常会导致CRISPR-Cas位点的小插入和缺失（indel）。 诱导DSB。因此，CRISPR系统是创建基因敲除的有效工具， 定向插入仍然是一个挑战。有效的靶向插入将允许快速产生癌症 模型，也将为下一代过继性T细胞疗法铺平道路，其中嵌合抗原 CAR转基因可以插入到特定的基因组位点，从而提高它们摧毁癌症的能力 细胞由供体模板介导的同源性驱动修复（HDR）是目前引入精确修复的最佳方式。 CRISPR-Cas切割后的编辑和DNA插入，但相对于NHEJ，效率通常较低， 改进战略只取得了很小的进展。在这里，我提出了一种改进HDR的方法， 使染色质在CRISPR-Cas诱导的切割位点更容易接近，并装饰相邻的核小体 H3 K36 me 3，一种已知参与介导HDR的组蛋白标记。为此，我设计了一个Cas9- PRDM 9融合构建体。PRDM 9是一种甲基转移酶，它将H3 K4 me 3和H3 K36 me 3沉积到标记物上， 在减数分裂过程中的重组热点，也已被证明是一个先锋因子，使染色质 更容易进入下游流程。在初步实验中，这种新的融合结构显示出 2-与单独的Cas9相比，HDR：indel比率的倍数改善。通过直接改变染色质结构， 假设HDR水平可以增加，以进行精确的基因组编辑和靶向DNA插入， 不同的细胞类型，而不管预先存在的染色质结构。在第一个目标中，我计划研究如何 位点特异性染色质修饰介导CRISPR-Cas9切割后的DNA修复。我也会学习 通过Cas9-PRDM 9融合的染色质结构的直接重构是否可以改善同源性？ 定向修复（HDR）在第二个目标中，我将研究对HDR的影响，钝与交错削减介绍 不同的Cas 12家族核酸酶，包括新鉴定的超紧密CasΦ，以及是否融合了 这些具有PRDM 9的编辑器可以提高精确的基因组编辑。最后，我将研究如何将这些结合起来， 可以利用这些特性来改善细胞系和人原代T细胞中的大DNA插入（> 100 bp）， 细胞