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CRISPR文库构建

云舟生物专注于高度复杂的CRISPR文库设计，以满足各种大规模和小规模功能筛选的实验需求。CRISPR文库可以以E. coli菌株、质粒DNA或重组病毒的形式交付。我们使用下一代测序（NGS）全面验证定制文库，能让您真正了解所购买文库的组分和质量。

亮点

  • 高多样性： 我们的CRISPR文库拥有从数百至106的高度复杂度。
  • CRISPR文库设计经验丰富： 我们对于多种类型的CRISPR文库针对不同种类的筛选策略拥有丰富的设计经验，包括但不限于CRISPR KO（单gRNA或双gRNA）、CRISPRa、CRISPRi、单细胞CRISPR技术、CRISPR barcoding技术等。
  • 高度灵活的载体设计： 我们可根据您的需求为您设计合适的文库载体，使用不同的递送系统、启动子、标记基因、barcode等。
  • 高质量病毒包装： 我们可包装多种病毒系统，周期短、性价比高
  • 高覆盖率、高均一性： 我们设计的载体变体其文库可达>98%的覆盖率
服务详情
landing page workflow
价格与周期

表1 文库构建服务价格与周期

服务简述价格（CNY）周期
设计文库构建策略CRISPR载体骨架设计、gRNA序列设计免费1-4 天
文库质粒克隆（包括oligo合成和NGS验证）见表2。
文库的病毒包装点击此处了解更多病毒包装服务信息。文库质粒的病毒病毒包装价格是常规单质粒载体包装的1.5倍。
功能筛选样品的NGS测序分析包括细胞样品基因组的NGS文库制备、lllumina测序（>500×覆盖率）和数据分析。￥ 2,240/样本3-5 周

表2 文库复杂度与文库载体克隆价格

文库复杂度价格（CNY）周期
单gRNA
0 ~ 5×103￥ 16,100起5-8 周
5×103 ~ 104￥ 31,500起
104 ~ 5×104￥ 38,500起6-10 周
5×104 ~ 105￥ 52,500起
>1×105请咨询
双gRNA
0 ~ 5×103￥ 16,100起9-13 周
5×103 ~ 104￥ 45,500起
104 ~ 5×104￥ 56,000起
5×104 ~ 105￥ 70,000起
>105请咨询
技术详情
CRISPR 筛选策略

CRISPR文库是一种强大的工具，可用于针对特定通路、疾病、细胞对药物治疗的反应、发育过程、基因调控等编码或非编码区域的大规模或全基因组功能筛选。表3展示了常用的CRISPR筛选方法并进行了比较。

表3 CRISPR筛选方法比较

筛选策略机制靶向区域应用
CRISPR KO野生型Cas9或Cas9核酸内切酶切割DNA。当细胞试图修复DNA断裂时，会在靶位点引入随机突变。主要为编码区域主要用于编码基因的功能筛选
CRISPRa酶切活性失活的突变型Cas9（dCas9）融合转录激活因子（VP64），结合靶位点调控激活基因表达。通常为启动子区域或者其它非编码调控区域编码基因的功能获得型筛选、非编码区域的调控功能筛选
CRISPRi酶切活性失活的突变型Cas9（dCas9）融合转录抑制因子（KRAB），结合靶位点调控抑制基因表达。通常为启动子区域或者其它非编码调控区域编码基因的功能缺失型筛选、非编码区域的调控功能筛选
实验数据
Complexity, coverage, and alignment rate of CRISPR libraries constructed by VectorBuilder

图1 云舟生物CRISPR文库的复杂度、覆盖率和序列比对的一致性。来自于云舟生物构建的CRISPR文库的数据以无偏好方式进行收集和分析。（A）文库复杂度范围从102到>105。（B）gRNA的覆盖率和读取的比对率均很高，表明我们的文库具有出色的覆盖率和低错误率。

Distribution of gRNA representation in a pooled library

图2 包含7.7×104个gRNA的文库的gRNA分布统计。gRNA read counts使用NGS库大小（一千万reads）进行标准化，并以log2尺度绘制。NGS测序深度为300×。

客户提供文库质粒

如果您需要使用您的文库质粒构建文库，请按照外来物品接收指南将质粒寄送给我们。请严格按照我们的指南来进行装运以免造成物品的任何延误和损坏。客户提供的所有材料均需经过QC检测，每一份材料附加检测费用。请注意，客户提供物品接收并通过QC之后生产才正式开始。对于使用客户文库质粒构建的文库，我们无法保证文库的多样性和均一性。

文档

使用说明书

Pooled Lentivirus Libraries for In Vitro Screening

常见问题解答

精选文献
Partial view-Genetic and pharmacological modulation of DNA mismatch repair heterogeneous tumors promotes immune surveillance
Genetic and pharmacological modulation of DNA mismatch repair heterogeneous tumors promotes immune surveillance
Vito Amodio, et al.
Cancer Cell, 2023, doi: 10.1016/j.ccell.2022.12.003
使用的云舟生物产品或服务：
Abstract: Patients affected by colorectal cancer (CRC) with DNA mismatch repair deficiency (MMRd), often respond to immune checkpoint blockade therapies, while those with mismatch repair-proficient (MMRp) tumors generally do not. Interestingly, a subset of MMRp CRCs contains variable fractions of MMRd cells, but it is unknown how their presence impacts immune surveillance. We asked whether modulation of the MMRd fraction in MMR heterogeneous tumors acts as an endogenous cancer vaccine by promoting immune surveillance. To test this hypothesis, we use isogenic MMRp (Mlh1+/+) and MMRd (Mlh1-/-) mouse CRC cells. MMRp/MMRd cells mixed at different ratios are injected in immunocompetent mice and tumor rejection is observed when at least 50% of cells are MMRd. To enrich the MMRd fraction, MMRp/MMRd tumors are treated with 6-thioguanine, which leads to tumor rejection. These results suggest that genetic and pharmacological modulation of the DNA mismatch repair machinery potentiate the immunogenicity of MMR heterogeneous tumors.
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Partial view-Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs
Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs
Núria Bosch-Guiteras, et al.
Genome Research, 2021, doi: 10.1101/gr.265736.120
使用的云舟生物产品或服务：
Abstract: CRISPR-Cas9 deletion (CRISPR-del) is the leading approach for eliminating DNA from mammalian cells and underpins a variety of genome-editing applications. Target DNA, defined by a pair of double-strand breaks (DSBs), is removed during nonhomologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and false negative results. By using an endogenous reporter system, we show that repression of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)—an early step in NHEJ—yields substantial increases in DNA deletion. This is observed across diverse cell lines, gene delivery methods, commercial inhibitors, and guide RNAs, including those that otherwise display negligible activity. We further show that DNA-PKcs inhibition can be used to boost the sensitivity of pooled functional screens and detect true-positive hits that would otherwise be overlooked. Thus, delaying the kinetics of NHEJ relative to DSB formation is a simple and effective means of enhancing CRISPR-deletion.
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Partial view-A Single-Cell Transcriptomics CRISPR-Activation Screen Identifies Epigenetic Regulators of the Zygotic Genome Activation Program
A Single-Cell Transcriptomics CRISPR-Activation Screen Identifies Epigenetic Regulators of the Zygotic Genome Activation Program
Celia Alda-Catalinas, et al.
Cell Systems, 2020, doi: 10.1016/j.cels.2020.06.004
使用的云舟生物产品或服务：
Abstract: Zygotic genome activation (ZGA) is an essential transcriptional event in embryonic development that coincides with extensive epigenetic reprogramming. Complex manipulation techniques and maternal stores of proteins preclude large-scale functional screens for ZGA regulators within early embryos. Here, we combined pooled CRISPR activation (CRISPRa) with single-cell transcriptomics to identify regulators of ZGA-like transcription in mouse embryonic stem cells, which serve as a tractable, in vitro proxy of early mouse embryos. Using multi-omics factor analysis (MOFA+) applied to ~200,000 single-cell transcriptomes comprising 230 CRISPRa perturbations, we characterized molecular signatures of ZGA and uncovered 24 factors that promote a ZGA-like response. Follow-up assays validated top screen hits, including the DNA-binding protein Dppa2, the chromatin remodeler Smarca5, and the transcription factor Patz1, and functional experiments revealed that Smarca5’s regulation of ZGA-like transcription is dependent on Dppa2. Together, our single-cell transcriptomic profiling of CRISPRa-perturbed cells provides both system-level and molecular insights into the mechanisms that orchestrate ZGA.
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