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CRISPR

CRISPR

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...

RNA Editing

RNA Editing

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...

What is Genetic Engineering?

What is Genetic Engineering?

Conservative Site-specific Recombination and Phase Variation

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...

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Related Experiment Video

Updated: May 21, 2025

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Published on: May 25, 2018

Genome editing with programmable base editors in human cells.

Nicola R B Osgood¹, Natalie M Zawalick¹, Courtney B Sawyer²

¹Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, United States.

Methods in Enzymology

|March 22, 2025

Summary

This summary is machine-generated.

This guide simplifies selecting base editors and designing guide RNAs (gRNAs) for precise genome editing. It offers practical methods for generating gRNA plasmids, cell transfection, and assessing editing efficiency, aiding researchers in this rapidly evolving field.

Keywords:

Adenine Base Editor (ABE)Base editing Cytosine Base Editor (CBE)Electroporation Fluorescence-activated cell sorting (FACS)Guide RNA (gRNA) design High throughput sequencing (HTS)IPSC base editing Isogenic cell line generation Next generation sequencing (NGS)Single-cell sorting Transfection gRNA synthesis

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Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

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Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

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Published on: February 28, 2021

Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

Genome editing technologies have rapidly advanced, expanding the toolkit for genetic engineering.
Base editors offer a precise method for introducing single-nucleotide changes without inducing double-strand DNA breaks.

Purpose of the Study:

To provide clear guidance on selecting appropriate base editors for specific genome engineering needs.
To detail the design and generation of effective guide RNAs (gRNAs) for base editing applications.
To offer practical protocols for base editing in mammalian cells, including transfection and efficiency evaluation.

Main Methods:

Selection criteria for various base editor systems.
Guide RNA (gRNA) design principles and plasmid generation protocols.
Mammalian cell transfection techniques and base editing efficiency assessment methods.

Main Results:

A structured approach to choosing the right base editor based on experimental requirements.
Detailed protocols for gRNA construction and delivery.
Methods for optimizing transfection and accurately measuring base editing outcomes.

Conclusions:

This resource aims to demystify base editor selection and application for researchers.
Effective gRNA design and optimized experimental procedures are crucial for successful base editing.
The guide provides practical solutions and troubleshooting advice for common challenges in base editing experiments.