(f)利用CRISPR-Tag系统同时显示mCherry-LMNA和LMNA基因座的代表性图像。(g)LMNA基因座标记效率的量化。 图7 将CRISPR-Tag隐藏在内含子中时,蛋白质表达保持正常(Chen et al., 2018)。(a)CRISPR-Tag_v2设计示意图。每个重复包含三个CRISPR靶向位点(TS5、TS6和TS7)。(b)CRISPR-Tag在mCherry内含子中的定位示意...
The lack of efficient tools to image non-repetitive genes in living cells has limited our ability to explore the functional impact of the spatiotemporal dynamics of such genes. Here, we addressed this issue by developing a CRISPR-Tag system using one to
检测TCRP1敲除效果的PCR引物分别是:5′-TGTCTGTGGGATAGTGGGAC-3′(TCRP1-DNA-F)和5′-CGTTAGGAGGAGGAGCAAGT-3′(TCRP1-DNA-R),取单一条带的PCR产物进行Sanger测序验证,初步验证成功后提取蛋白进行Western blot检测,选择稳定敲除的单...
Add a GFP or RFP tag to a target gene without the need for cloning Design all necessary tools for precise tagging, SNP, amino acid, or knockout changes Order all materials with one click from a single source with confidence that they will work together seamlessly...
. . . ## AGGTAGAG . . . . . ## GAGCTCCT . 1 . 1 . The designOpsLibrary allows users to perform a complete end-to-end library design; see ?designOpsLibrary for documentation. For more information, please see the following tutorial: Design for OPS Design of gRNA pairs with the ...
5’ACACCGNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTT3’ 反向序列 3’TGTGGCNNNNNNNNNNNNNNNNNNNCAAAATCTCGATCTTTATCGTTCAATTTTATTCCGATCAGGCAA5’ 插入片段的合成 用水将寡核苷酸稀释为 100 μM。按以下体系配制退火反应体系:
5'-TCCTAGGTTGAAGATAACCCACATAATAAG-3'；针对Orf1ab引物对 Orf1ab-RPA-Forward_v1：5’-GAATAATATACGACTACGGAGGAGTGTGAGATAGACATAT ActAAAC-3’；Orf1ab-RPA-Reverse_v1:5'-TAGTAAGACTAGAATTGTCTACATAAGCAGC-3'；用于检测S基因的LwaCas13acrRNA S-crRNA_v1：5’-GuuuAgAccCCAAACAGGAGGACUCAAACAGCGACCAC...
CTTTATATATCTTGTGGAAAGGACGAAACACCG-TTCGCACGATTGCACCTTGG-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG 在sgRNA的两端设计合适的同源臂序列,方便后续的PCR扩增及无缝克隆至目标载体。 | sgRNA pool构建步骤 a. 载体处理 通常需要先抽提获得高纯度的目的质粒载体,这样才可以保证后续线性化载体后的纯度能够符合构建文库的要求...
本研究通过BreakTag方法全面解析了Cas9诱导的双链断裂及其末端结构,揭示了错位切割在校正病理性单核苷酸缺失中的重要作用。实验结果表明,约35%的SpCas9 DSB是错位的,这种切割类型受到DNA:gRNA互补性和工程化Cas9变体的显著影响。通过选择合适的Cas9变体和优化gRNA设计,可以实现高效、精准的基因修复,为未来的基因治疗提供了...
Three-component repurposed technology for enhanced expression: highly accumulable transcriptional activators via branched tag arrays. CRISPR J. 1, 337–347 (2018). Article CAS PubMed PubMed Central Google Scholar Abudayyeh, O. O. et al. C2c2 is a single-component programmable RNA-guided RNA...