Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

165
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...
165
CRISPR01:59

CRISPR

52.7K
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...
52.7K
Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

6.1K
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...
6.1K
CRISPR and crRNAs02:53

CRISPR and crRNAs

17.3K
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.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
17.3K
Homologous Recombination02:31

Homologous Recombination

50.9K
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...
50.9K

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

GLIS3 marks a neural-like progenitor cell state that drives metastasis in pancreatic ductal adenocarcinoma.

Cell reports·2026
Same author

Pre-training genomic language model with variants for better modeling functional genomics.

NPJ artificial intelligence·2026
Same author

The discovery of 5mC-selective deaminases and their application to ultra-sensitive direct sequencing of methylated sites at base resolution.

Molecular cell·2026
Same author

Distinct Malignant Cell States and Myeloid Glutamate Signaling Associated with Aggressive Pancreatic Neuroendocrine Tumors.

Clinical cancer research : an official journal of the American Association for Cancer Research·2026
Same author

EPInformer: scalable and integrative prediction of gene expression from promoter-enhancer sequences with multimodal epigenomic profiles.

Nature communications·2026
Same author

Immune evasive DNA donors and recombinases license kilobase-scale writing.

Nature·2026
Same journal

Author Correction: Improved RNA base editing with guide RNAs mimicking highly edited endogenous ADAR substrates.

Nature biotechnology·2026
Same journal

Unlocking the chemical potential of filamentous fungi using prime editing.

Nature biotechnology·2026
Same journal

A genome-scale CRISPRi perturbation atlas of human induced pluripotent stem cells.

Nature biotechnology·2026
Same journal

Prime editing for precise genome engineering and modulation of fungal metabolism.

Nature biotechnology·2026
Same journal

Retargeted serine integrases for one-step, precise integration of large DNA sequences in human cells.

Nature biotechnology·2026
Same journal

A retargeted recombinase for precise insertion of large DNA.

Nature biotechnology·2026
See all related articles

Related Experiment Video

Updated: Aug 26, 2025

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

819

Precise DNA cleavage using CRISPR-SpRYgests.

Kathleen A Christie1,2,3, Jimmy A Guo1,2,4, Rachel A Silverstein1,2,4

  • 1Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA.

Nature Biotechnology
|October 6, 2022
PubMed
Summary
This summary is machine-generated.

A new CRISPR-Cas9 variant, SpRY, acts as a PAMless DNA cleavage tool for precise molecular cloning. SpRY digests (SpRYgests) enable DNA breaks at virtually any sequence, enhancing cloning applications.

More Related Videos

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
11:35

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Published on: June 16, 2017

12.7K
Substrate Generation for Endonucleases of CRISPR/Cas Systems
11:53

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Published on: September 8, 2012

27.5K

Related Experiment Videos

Last Updated: Aug 26, 2025

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

819
Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
11:35

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Published on: June 16, 2017

12.7K
Substrate Generation for Endonucleases of CRISPR/Cas Systems
11:53

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Published on: September 8, 2012

27.5K

Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genomic Engineering

Background:

  • Current DNA cleavage methods like restriction enzymes and wild-type CRISPR-Cas9 have limitations due to specific motif or protospacer adjacent motif (PAM) requirements.
  • Precise DNA cleavage adjacent to target bases is crucial for advanced molecular cloning and DNA engineering applications.

Purpose of the Study:

  • To evaluate the utility of the near-PAMless SpCas9 variant, SpRY, as a universal DNA cleavage tool for molecular cloning.
  • To demonstrate the effectiveness of SpRY-mediated DNA digests (SpRYgests) for precise DNA breaks in various cloning workflows.

Main Methods:

  • In vitro DNA cleavage assays using SpRY with over 130 guide RNAs (gRNAs) across diverse PAM sequences.
  • Application of SpRYgests in multiple DNA engineering and molecular cloning workflows.
  • Optimization of a one-pot gRNA synthesis protocol for streamlined SpRYgest implementation.

Main Results:

  • SpRY demonstrated PAMless DNA cleavage in vitro, capable of cutting DNA at practically any sequence.
  • SpRY successfully cleaved DNA at sites refractory to wild-type SpCas9.
  • SpRYgests improved the precision of several cloning workflows, including those not feasible with traditional methods.

Conclusions:

  • SpRY functions as a versatile, PAMless DNA cleavage tool for in vitro applications.
  • SpRYgests offer enhanced precision and broader applicability in DNA engineering and molecular cloning.
  • Optimized gRNA synthesis further facilitates the adoption of SpRYgests in research.