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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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CRISPR01:59

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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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CRISPR and crRNAs02:53

CRISPR and crRNAs

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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
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Homologous Recombination02:31

Homologous Recombination

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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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Related Experiment Video

Updated: Jul 13, 2025

Genome Editing in Mammalian Cell Lines using CRISPR-Cas
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Efficient genome editing in grapevine using CRISPR/LbCas12a system.

Chong Ren1,2,3, Elias Kirabi Gathunga1,2,3, Xue Li1,2,3

  • 1State Key Laboratory of Plant Diversity and Specialty Crops, Beijing Key Laboratory of Grape Sciences and Enology, Institute of Botany, the Chinese Academy of Sciences, Beijing, 100093, PR China.

Molecular Horticulture
|October 18, 2023
PubMed
Summary
This summary is machine-generated.

The CRISPR/Cas12a system, specifically LbCas12a, is now effective for grapevine genome engineering. This system successfully induced mutations in target genes, with heat treatment enhancing efficiency and truncated crRNAs showing promise for precise gene editing.

Keywords:
CRISPRFlavonoidLbCas12aMultiplex editingVitis viniferacrRNA length

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Area of Science:

  • Plant Biotechnology
  • Molecular Biology
  • Genetics

Background:

  • The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas12a system offers powerful genome engineering capabilities in various plants.
  • Grapevine (Vitis vinifera) has not yet benefited from CRISPR/Cas12a applications for genetic modification.

Purpose of the Study:

  • To develop and validate the CRISPR/Cas12a system, specifically Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a), for targeted mutagenesis in grapevine cells.
  • To investigate the impact of gene knockout on flavonoid accumulation and optimize editing efficiency through environmental and molecular modifications.

Main Methods:

  • CRISPR/LbCas12a was employed to target the tonoplastic monosaccharide transporter1 (TMT1) and dihydroflavonol-4-reductase 1 (DFR1) genes in grapevine 41B cells.
  • Mutagenesis was induced, and the effects of heat treatment (34°C) on editing efficiency were evaluated.
  • The influence of crRNA sequence length and truncated crRNAs (trucrRNAs) on genome editing outcomes was analyzed.

Main Results:

  • Targeted mutagenesis was successfully achieved in grapevine 41B cells by knocking out the DFR1 gene, leading to altered flavonoid accumulation.
  • Heat treatment significantly improved CRISPR/LbCas12a editing efficiencies for TMT1, increasing them from 35.3% to 44.6% and 29.9% to 37.3%.
  • crRNA sequence length was identified as a key factor for editing efficiency, with 20 nt trucrRNAs proving as effective as 24 nt crRNAs, while shorter sequences (≤18 nt) were less effective.

Conclusions:

  • The CRISPR/LbCas12a system is a viable and effective tool for genome engineering in grapevine.
  • Optimizing conditions like heat treatment and crRNA design, including the use of trucrRNAs, enhances the precision and efficiency of grapevine gene editing.
  • This study paves the way for advanced genetic manipulation and crop improvement in grapevine using CRISPR/LbCas12a technology.