Screening and identifying of compound for promoting gene editing and application of compound
A gene editing and compound technology, applied in the field of biology, can solve the problem of unsatisfactory gene editing efficiency of CRISPR-Cpf1
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0183] Efficient construction of knockout cell lines using CRISPR-Cpf1 in human pluripotent stem cells
[0184] In order to use the CRISPR-Cpf1 gene editing system in human pluripotent stem cells, a crRNA expression plasmid driven by the U6 promoter was constructed ( Figure 4 a and 4b).
[0185] Several genes of interest were picked, including ALKBH1 and CLEC16A. ALKBH1 is a tRNA demethylase, and CLEC16A plays an important role in the occurrence of diabetes.
[0186] To construct gene-specific crRNA plasmids, a set of crRNAs specifically targeting these genes was designed ( figure 1 b and Table 1).
[0187] 【Table 1】
[0188] Construction of nucleic acid sequences expressing crRNA plasmids
[0189]
[0190]
[0191] PCR primers for constructing plasmids
[0192]
[0193]
[0194] PCR primers for genotyping and sequencing
[0195]
[0196] PCR primers for T7EI experiments
[0197]
[0198] Primers for RFLP experiments
[0199]
[0200] Primers for...
Embodiment 2
[0215] Screening of small molecules that can significantly promote CRISPR-Cpf1-mediated HDR in hPSCs
[0216] In this example, in order to test the ability of CRISPR-Cpf1 for gene insertion, three plasmids were transferred into hPSCs by electroporation: one plasmid expresses Cpf1, one contains a specific crRNA targeting OCT4, and contains the eGFP reporter gene and puromycin (Puro) resistant HDR template plasmid ( Figure 8 a). After electroporation, hPSCs were first cultured with ordinary medium for two days, and then puromycin was added to the medium for 3-4 days. It was observed that the efficiency of genome repair by HDR approach is relatively low and needs to be further improved.
[0217] In addition, SCR7 compounds known to increase the efficiency of CRISPR-Cas9-mediated gene editing were used as control compounds.
[0218]
[0219] result:
[0220] Experiments showed that SCR7, a small molecule known to promote CRISPR-Cas9-mediated gene knock-in, had no significant...
Embodiment 3
[0223] Chemical Small Molecule Screening
[0224] In this example, hundreds of candidate compounds were screened and tested one by one.
[0225] In this example, in order to improve the efficiency of CRISPR-Cpf1-mediated gene editing in hPSCs, large-scale compound screening was performed using the OCT4-eGFP knock-in selection system with puromycin ( figure 2 a).
[0226] Specifically, three plasmids were transferred into hPSCs by electroporation: one expressing Cpf1, one containing a specific crRNA targeting OCT4, and an HDR template plasmid containing an eGFP reporter gene and puromycin (Puro) resistance ( Figure 8 a).
[0227] For each candidate compound, 100 μL of cell culture medium and 0.2 μL of screened small molecules (1:1000) were added to each well of a 48-well plate before electroporation. After cell electroporation, 1x10 6 The cells were mixed in 4.8mL hPSC medium supplemented with Thiazovivin small molecules, and seeded in a 48-well plate. The cells were cul...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More - R&D
- Intellectual Property
- Life Sciences
- Materials
- Tech Scout
- Unparalleled Data Quality
- Higher Quality Content
- 60% Fewer Hallucinations
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com