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RNA editing is a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the...
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Genomics is the science of genomes: it is the study of all the genetic material of an organism. In humans, the genome consists of information carried in 23 pairs of chromosomes in the nucleus, as well as mitochondrial DNA. In genomics, both coding and non-coding DNA is sequenced and analyzed. Genomics allows a better understanding of all living things, their evolution, and their diversity. It has a myriad of uses: for example, to build phylogenetic trees, to improve productivity and...
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Diploid organisms inherit genetic material through chromosomes from both parents. Copies of the same gene are known as alleles. In most cases, both alleles are simultaneously expressed and allow various cellular processes to function optimally. If one of the alleles is missing or mutated, the expression of the other allele can compensate; however, this is not true for all genes.
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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs.

Sachiko Okamoto1, Yasunori Amaishi1, Izumi Maki1

  • 1CDM Center, Takara Bio Inc. Nojihigashi 7-4-38, Kusatsu, Shiga, 525-0058, Japan.

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|March 20, 2019
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Optimizing single-stranded oligonucleotide (ssODN) design significantly enhances gene knock-in efficiency for genome editing. Using Cas9 protein/sgRNA ribonucleoprotein complexes (Cas9-RNPs) further improves accuracy by minimizing unwanted DNA re-cutting.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Genome editing with engineered nucleases is crucial in research and therapeutics.
  • Homologous recombination (HR) enables precise gene editing but often suffers from low efficiency, especially for knock-ins and single-base substitutions.

Purpose of the Study:

  • To optimize single-stranded oligonucleotide (ssODN) design for improved knock-in efficiency in genome editing.
  • To evaluate the impact of ssODN parameters like blocking mutations, orientation, size, and homology arm length.
  • To compare the efficiency of different delivery methods for Cas9 and sgRNA, including ribonucleoprotein complexes.

Main Methods:

  • Utilized reporter systems to detect single-base substitutions and assess knock-in efficiency.
  • Systematically varied ssODN design features (blocking mutation, orientation, size, homology arm length).
  • Investigated various delivery forms and methods for Cas9 and sgRNA, including Cas9 protein/sgRNA ribonucleoprotein complexes (Cas9-RNPs).

Main Results:

  • Optimized ssODN designs with blocking mutations demonstrated significantly higher knock-in efficiencies compared to those without.
  • Cas9-RNPs effectively reduced the re-cutting of edited genomic sites, enhancing editing precision.
  • Specific ssODN design parameters were identified as critical for maximizing knock-in success.

Conclusions:

  • Strategic design of ssODNs is essential for boosting the efficiency of gene knock-in via homologous recombination.
  • Cas9-RNPs represent a superior delivery method for precise genome editing by minimizing secondary modifications.
  • These findings provide valuable insights for enhancing genome editing strategies in various applications.