Related Experiment Video

Updated: Dec 27, 2025

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors
09:22

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

Published on: February 28, 2021

5.9K

Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a

Eszter Tóth1, Éva Varga1,2,3, Péter István Kulcsár1,2,3

  • 1Institute of Enzymology, Research Centre of Natural Sciences of the Hungarian Academy of Sciences, Budapest H-1117, Hungary.

Nucleic Acids Research
|February 29, 2020
PubMed

Insights

Researchers engineered new Cas12a variants to overcome protospacer adjacent motif (PAM) limitations in genome editing. These modified Cas12a nucleases exhibit broader PAM compatibility, enhancing gene editing applications in various cells.

Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetics

Background:

  • The CRISPR-Cas12a system is crucial for genome engineering.
  • Its application is restricted by the need for a long TTTV protospacer adjacent motif (PAM).

Purpose of the Study:

  • To develop Cas12a variants with relaxed PAM constraints.
  • To enhance genome editing and transcriptome modulation efficiency.

Main Methods:

  • Generated and combined mutations from existing Cas12a variants (RR and RVR) with altered PAM specificities.
  • Selected LbCas12a-RVRR for in-depth PAM preference characterization in mammalian cells using plasmid-based assays.
  • Investigated the D156R mutation in improved LbCas12a (impLbCas12a) for enhanced activity.

Main Results:

  • Developed novel Cas12a variants active in mammalian and plant cells with loosened PAM requirements.
  • LbCas12a-RVRR demonstrated a consensus PAM of TNTN, including TACV, TTCV, CTCV, and CCCV.
  • The D156R mutation in impLbCas12a further increased PAM-dependent activity.
  • Overlapping PAM preferences of impLbCas12a and enAsCas12a enhance genome editing efficiency.

Conclusions:

  • Engineered Cas12a variants significantly expand PAM compatibility for genome engineering.
  • These variants, particularly impLbCas12a and enAsCas12a, offer complementary functionalities for advanced gene editing and transcriptome modulation.

More Related Videos

Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e07:31

Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e

Published on: February 17, 2023

1.5K
Genome Editing in Mammalian Cell Lines using CRISPR-Cas07:56

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Published on: April 11, 2019

23.0K

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...
1.5K
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...
6.5K
Long-patch Base Excision Repair01:02

Long-patch Base Excision Repair

Since the discovery of the two BER pathways, there has been a debate about how a cell chooses one pathway over the other and the factors determining this selection. Numerous in vitro experiments have pointed out multiple determinants for the sub-pathway selection. These are:
7.7K