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Related Concept Videos

Homologous Recombination02:31

Homologous Recombination

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...
Fixing Double-strand Breaks02:04

Fixing Double-strand Breaks

The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the back-up in case the other strand is damaged. Second, the double-helical structure can be wrapped around proteins called histones to form nucleosomes, which can then be tightly wound to form chromosomes. This way, DNA chains up to 2 inches long can be contained within microscopic structures in a cell. A double-stranded break not only damages...
Fixing Double-strand Breaks02:04

Fixing Double-strand Breaks

The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the back-up in case the other strand is damaged. Second, the double-helical structure can be wrapped around proteins called histones to form nucleosomes, which can then be tightly wound to form chromosomes. This way, DNA chains up to 2 inches long can be contained within microscopic structures in a cell. A double-stranded break not only damages...
Base-pairing and DNA Repair02:27

Base-pairing and DNA Repair

Erwin Chargaff’s rules on DNA equivalence paved the way for the discovery of base pairing in DNA. Chargaff’s rules state that in a double-stranded DNA molecule,
DNA Damage can Stall the Cell Cycle02:36

DNA Damage can Stall the Cell Cycle

In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...
DNA Damage Can Stall the Cell Cycle02:36

DNA Damage Can Stall the Cell Cycle

In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...

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Related Experiment Video

Updated: May 19, 2026

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells
10:07

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Published on: August 25, 2017

Parallel Activation and Interference CRISPR (PAIR) with Sequencing Uncovers DNA Repair Networks Guiding Precision

Chen Chang1,2, Dailin Gan3, Lu Diao1,2

  • 1Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.

Biorxiv : the Preprint Server for Biology
|May 18, 2026
PubMed
Summary

We developed a bidirectional CRISPR system (PAIR) to study opposing gene interactions in DNA repair. This approach enhances gene editing precision and improves CAR T cell engineering by manipulating cellular repair pathways.

Keywords:
CRISPR screenCRISPR/Cas13dCRISPRaDNA damage response (DDR)Genome engineeringNon-viral CAR TSingle cell perturb-seq

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CIRCLE-Seq for Interrogation of Off-Target Gene Editing
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CIRCLE-Seq for Interrogation of Off-Target Gene Editing

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Last Updated: May 19, 2026

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells
10:07

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Published on: August 25, 2017

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter
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CIRCLE-Seq for Interrogation of Off-Target Gene Editing
08:23

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Published on: November 1, 2024

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Cellular homeostasis relies on balanced regulatory pathways, but traditional genetic screens often overlook bidirectional gene-gene interactions crucial for complex processes like DNA damage response (DDR).
  • Understanding these interactions is key to deciphering cellular phenotypes and improving biotechnological applications.

Purpose of the Study:

  • To introduce PAIR (Parallel Activation and Interference CRISPR), a novel platform for simultaneous gene activation and suppression within single cells.
  • To explore bidirectional gene-gene interactions in DNA double-strand break (DSB) repair pathways.
  • To enhance CRISPR-based gene editing precision and advance chimeric antigen receptor (CAR) T cell engineering.

Main Methods:

  • Development of the PAIR platform combining CRISPR activation (CRISPRa) and Cas13d RNA knockdown for bidirectional gene perturbation.
  • Application of PAIR in a CRISPR/Cas9 induced DSB repair screen to map gene interactions.
  • Integration of PAIR with single-cell transcriptomics to analyze cellular responses.
  • Development of an mRNA-based strategy for non-viral CAR T cell engineering using PAIR principles.

Main Results:

  • PAIR successfully mapped gene-gene interactions in competing DNA repair pathways, identifying synergistic perturbations like NBN activation with end-joining factor suppression.
  • These perturbations shifted repair outcomes towards homology-directed repair (HDR), improving CRISPR gene editing accuracy.
  • NBN activation was shown to induce inflammatory responses, which were modulated by co-suppressing end-joining factors, revealing previously hidden transcriptional states.
  • The mRNA-based strategy enhanced CAR T cell engineering by creating a transient HDR-favored state, improving CAR knock-in efficiency.

Conclusions:

  • The PAIR system offers a versatile framework for investigating opposing regulatory networks and uncovering complex cellular states.
  • Bidirectional perturbation is essential for a comprehensive understanding of gene interactions and cellular phenotypes.
  • PAIR technology has significant implications for improving gene editing technologies and cell-based therapies, such as CAR T cells.