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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...

CRISPR and crRNAs

CRISPR and crRNAs

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.
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Reproductive Cloning

Reproductive Cloning

Reproductive cloning is the process of producing a genetically identical copy—a clone—of an entire organism. While clones can be produced by splitting an early embryo—similar to what happens naturally with identical twins—cloning of adult animals is usually done by a process called somatic cell nuclear transfer (SCNT).
Somatic Cell Nuclear Transfer
In SCNT, an egg cell is taken from an animal and its nucleus is removed, creating an enucleated egg. Then a somatic...

RNA Interference

RNA Interference

RNA interference (RNAi) is a process in which a small non-coding RNA molecule blocks the post-transcriptional expression of a gene by binding to its messenger RNA (mRNA) and preventing the protein from being translated.
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Eukaryotic RNA Polymerases

Eukaryotic RNA Polymerases

RNA Polymerase (RNAP) is conserved in all animals, with bacterial, archaeal, and eukaryotic RNAPs sharing significant sequence, structural, and functional similarities. Among the three eukaryotic RNAPs, RNA Polymerase II is most similar to bacterial RNAP in terms of both structural organization and folding topologies of the enzyme subunits. However, these similarities are not reflected in their mechanism of action.
All three eukaryotic RNAPs require specific transcription factors, of which the...

Cloning of Dolly the Sheep

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The first successfully cloned mammal was Dolly, a sheep, born on 5th July 1996 at Roslin Institute, Scotland. The cloned sheep was named after the American singer Dolly Parton. Dolly lived for seven years and died of respiratory complications, which is speculated to be due to the actual age of her DNA. Because the DNA in cloned cells belongs to an older individual, the cloned individual’s life expectancy may be affected. Indeed, analysis of Dolly’s DNA revealed shorter...

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Related Experiment Video

Updated: Feb 3, 2026

CRISPR Guide RNA Cloning for Mammalian Systems

CRISPR Guide RNA Cloning for Mammalian Systems

Published on: October 2, 2018

CRISPR Guide RNA Cloning for Mammalian Systems.

Sathiji Nageshwaran¹, Alejandro Chavez², Nan Cher Yeo¹

¹Wyss Institute for Biologically Inspired Engineering, Harvard University; Department of Genetics, Harvard Medical School.

Journal of Visualized Experiments : Jove

|October 23, 2018

Summary

This summary is machine-generated.

This study details a cost-effective method for generating Cas9 guide RNAs (gRNAs) using BsmBI restriction enzyme and compatible vectors. The protocol is ideal for single or paired gRNA cloning, not large libraries.

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CRISPR Guide RNA Cloning for Mammalian Systems

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Area of Science:

Molecular Biology
Biotechnology
Gene Editing

Background:

CRISPR-Cas9 technology relies on guide RNAs (gRNAs) for target specificity.
Efficient and cost-effective gRNA generation is crucial for widespread adoption of gene editing tools.

Purpose of the Study:

To present a streamlined protocol for generating Cas9-associated guide RNAs (gRNAs).
To offer two alternative cloning strategies for gRNA expression vectors.

Main Methods:

Utilized the Type IIS restriction enzyme BsmBI for gRNA cloning.
Employed a set of compatible vectors for gRNA expression.
Sanger sequencing was used for vector validation.

Main Results:

Developed efficient and cost-effective methods for single or paired gRNA generation.
The protocol requires standard molecular biology equipment and reagents.
Demonstrated the applicability of the strategy to various organisms with appropriate vectors.

Conclusions:

The described protocol provides a practical approach for generating Cas9 gRNAs for individual applications.
The method is not suitable for large-scale gRNA library synthesis.
The general strategy is adaptable for different organisms.