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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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RNA Editing02:23

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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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Genomics02:02

Genomics

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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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Genomic Imprinting and Inheritance02:30

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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.
The expression of some genes depends on which parent passed the gene to the offspring, through a phenomenon known as...
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Genome Size and the Evolution of New Genes03:21

Genome Size and the Evolution of New Genes

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While every living organism has a genome of some kind (be it RNA, or DNA), there is considerable variation in the sizes of these blueprints. One major factor that impacts genome size is whether the organism is prokaryotic or eukaryotic. In prokaryotes, the genome contains little to no non-coding sequence, such that genes are tightly clustered in groups or operons sequentially along the chromosome. Conversely, the genes in eukaryotes are punctuated by long stretches of non-coding sequence.
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Cis-regulatory Sequences02:02

Cis-regulatory Sequences

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Cis-regulatory sequences are short fragments of non-coding DNA that are present on the same chromosomes as the genes that they regulate. These fragments serve as binding sites for transcriptional regulators, proteins that are responsible for controlling gene transcription and differential gene expression across cell types in eukaryotes. Cis-regulatory sequences can be close to the gene of interest or thousands of bases away in the DNA sequence; however, those sequences that are further away are...
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Updated: Jan 27, 2026

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources
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Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

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Optimizing genome editing strategy by primer-extension-mediated sequencing.

Jianhang Yin1,2, Mengzhu Liu1, Yang Liu1

  • 11The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking University, Beijing, 100871 China.

Cell Discovery
|April 3, 2019
PubMed
Summary
This summary is machine-generated.

Primer-extension-mediated sequencing (PEM-seq) comprehensively assesses genome editing tools. This method reveals CRISPR/Cas9 can cause large genomic alterations and helps select optimal genome editing strategies by evaluating both editing ability and specificity.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Efficient and precise genome editing is crucial for clinical applications and animal model development.
  • Engineered nucleases must exhibit high editing efficiency and low off-target activity for safe and effective use.

Purpose of the Study:

  • To introduce primer-extension-mediated sequencing (PEM-seq) for comprehensive assessment of engineered nucleases.
  • To evaluate the editing ability and specificity of various CRISPR-Cas9 systems and inhibitors.

Main Methods:

  • Development and application of primer-extension-mediated sequencing (PEM-seq) for high-throughput analysis.
  • Assessment of wild-type Cas9, Cas9 nickase, high-fidelity Cas9 variants (eCas9, FeCas9), and AcrIIA4 inhibitor.

Main Results:

  • PEM-seq identified chromosomal translocations and large deletions induced by CRISPR/Cas9.
  • Cas9 nickase showed reduced off-target activity but also decreased on-target cleavage.
  • High-fidelity Cas9 variants (eCas9, FeCas9) significantly lowered off-target activity without compromising editing efficiency.
  • AcrIIA4 inhibitor reduced Cas9 activity but was less effective at suppressing off-target loci compared to on-target sites.

Conclusions:

  • PEM-seq provides a comprehensive evaluation of engineered nucleases, surpassing methods that only detect off-target activity.
  • The findings aid in selecting superior genome editing strategies for specific genomic loci based on detailed nuclease performance.