Related Experiment Video

Updated: Oct 29, 2025

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
09:51

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Published on: May 25, 2018

34.7K

Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases

Moritz J Schmidt1, Ashish Gupta1, Christien Bednarski1

  • 1Bayer AG, Leverkusen, Germany.

Nature Communications
|July 10, 2021
PubMed

Insights

Researchers engineered smaller, highly effective CRISPR gene editing tools called synthetic RNA-guided nucleases (sRGNs). These novel Cas9 alternatives show promise for gene therapy, overcoming limitations of current systems for treating diseases.

Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Biotechnology

Background:

  • Streptococcus pyogenes Cas9 (SpyCas9) is a powerful gene editing tool but its large size poses delivery challenges for gene therapy.
  • Existing smaller Cas9 variants often exhibit reduced activity or specificity and recognize different PAM sequences.
  • There is a need for smaller, highly efficient, and specific Cas9 nucleases for therapeutic applications.

Purpose of the Study:

  • To investigate uncharacterized smaller Cas9 proteins as potential alternatives to SpyCas9.
  • To engineer novel synthetic RNA-guided nucleases (sRGNs) with enhanced editing efficiency and specificity.
  • To evaluate the therapeutic potential of sRGNs in vitro and in vivo for gene therapy applications.

Main Methods:

  • Screening of four uncharacterized smaller Cas9 proteins.
  • Protein engineering of identified Cas9 variants to create sRGNs.
  • In vitro and human cell line validation of sRGN editing efficiency and specificity.
  • In vivo delivery studies using mRNA lipid nanoparticles in mice and adeno-associated virus (AAV) vectors in non-human primates (NHPs).

Main Results:

  • Three novel smaller Cas9 enzymes were identified, utilizing a "GG" PAM sequence similar to SpyCas9.
  • Engineered sRGNs demonstrated superior in vitro and in human cell line editing efficiency and specificity compared to SpyCas9.
  • sRGN mRNA lipid nanoparticles showed efficient in vivo editing in mouse liver.
  • sRGNs, but not SpyCas9, were successfully packaged into all-in-one AAV vectors for robust in vivo editing in NHP retina photoreceptors.

Conclusions:

  • Engineered sRGNs represent a significant advancement over existing CRISPR nucleases, offering improved performance and delivery advantages.
  • These novel nucleases overcome key limitations of SpyCas9, paving the way for more effective gene therapies.
  • The successful in vivo application in NHP retina photoreceptors highlights the potential of sRGNs for treating genetic disorders affecting vision.

More Related Videos

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells11:35

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

Published on: June 16, 2017

12.9K
A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins08:37

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Published on: April 30, 2018

7.8K

Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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...
746
CRISPR01:59

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...
53.7K
Conservative Site-specific Recombination and Phase Variation02:53

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...
6.3K
CRISPR and crRNAs02:53

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.
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...
18.0K
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...
57.0K