PUBLICATION
A simple and rapid method for enzymatic synthesis of CRISPR-Cas9 sgRNA libraries
- Authors
- Yates, J.D., Russell, R.C., Barton, N.J., Yost, H.J., Hill, J.T.
- ID
- ZDB-PUB-210924-5
- Date
- 2021
- Source
- Nucleic acids research 49(22): e131 (Journal)
- Registered Authors
- Yost, H. Joseph
- Keywords
- none
- MeSH Terms
-
- Animals
- CRISPR-Associated Proteins
- CRISPR-Cas Systems*
- DNA Restriction Enzymes
- DNA-Directed DNA Polymerase
- Escherichia coli/genetics
- Fluorescent Dyes
- Genetic Techniques
- Genome
- Green Fluorescent Proteins
- Humans
- Myocardium/metabolism
- Oligonucleotides
- RNA/biosynthesis
- Transcriptome
- Zebrafish
- PubMed
- 34554233 Full text @ Nucleic Acids Res.
Citation
Yates, J.D., Russell, R.C., Barton, N.J., Yost, H.J., Hill, J.T. (2021) A simple and rapid method for enzymatic synthesis of CRISPR-Cas9 sgRNA libraries. Nucleic acids research. 49(22):e131.
Abstract
CRISPR-Cas9 sgRNA libraries have transformed functional genetic screening and have enabled several innovative methods that rely on simultaneously targeting numerous genetic loci. Such libraries could be used in a vast number of biological systems and in the development of new technologies, but library generation is hindered by the cost, time, and sequence data required for sgRNA library synthesis. Here, we describe a rapid enzymatic method for generating robust, variant-matched libraries from any source of cDNA in under 3 h. This method, which we have named SLALOM, utilizes a custom sgRNA scaffold sequence and a novel method for detaching oligonucleotides from solid supports by a strand displacing polymerase. With this method, we constructed libraries targeting the E. coli genome and the transcriptome of developing zebrafish hearts, demonstrating its ability to expand the reach of CRISPR technology and facilitate methods requiring custom libraries.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping