ArticleScience advances2016
A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism.
Article in Science advances, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed, 22 citations in OpenAlex.
- IgAim: cell surface Ig-aimed immune memory erasers for the therapy of autoimmune diseases and B leukemia.NAR molecular medicine · 2025Article
- Cross-contamination of CRISPR guides and other unrelated nucleotide sequences among commercial oligonucleotides.Nucleic acids research · 2024Article
- Review
- A new method to synthesize multiple gRNA libraries and functional mapping of mammalian H3K4me3 regions.Nucleic acids research · 2023Article
- miR-AB, a miRNA-based shRNA viral toolkit for multicolor-barcoded multiplex RNAi at a single-cell level.EMBO reports · 2022Article
- A simple and rapid method for enzymatic synthesis of CRISPR-Cas9 sgRNA libraries.Nucleic acids research · 2021Article
- Applying CRISPR Screen in Diabetes Research.Diabetes · 2021Review
- Development of a DNA double-strand break-free base editing tool inMetabolic engineering communications · 2020Article
- Unbiased and Tailored CRISPR/Cas gRNA Libraries by SynthesizingCovalently-closed-circular (3Cs) DNA.Bio-protocol · 2020Article
- Designing custom CRISPR libraries for hypothesis-driven drug target discovery.Computational and structural biotechnology journal · 2020Review
- Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome.eLife · 2019Article
- ESCO1/2's roles in chromosome structure and interphase chromatin organization.Genes & development · 2017Article
- Dense sgRNA Library Construction Using a Molecular Chipper Approach.Bio-protocol · 2017Article
- A Method to Convert mRNA into a Guide RNA (gRNA) Library without Requiring Previous Bioinformatics Knowledge of the Organism.Bio-protocol · 2017Article
- A double-strand break can trigger immunoglobulin gene conversion.Nucleic acids research · 2017Article
Corrections and comments
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Authors and funding
1 author at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system is a powerful tool for genome editing that can be used to construct a guide RNA (gRNA) library for genetic screening. For gRNA design, one must know the sequence of the 20-mer flanking the protospacer adjacent motif (PAM), which seriously impedes experimentally making gRNA. I describe a method to construct a gRNA library via molecular biology techniques without relying on bioinformatics. Briefly, one synthesizes complementary DNA from the mRNA sequence using a semi-random primer containing a PAM complementary sequence and then cuts out the 20-mer adjacent to the PAM using type IIS and type III restriction enzymes to create a gRNA library. The described approach does not require prior knowledge about the target DNA sequences, making it applicable to any species.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.