Journal
BIO-PROTOCOL
Volume 10, Issue 20, Pages -Publisher
BIO-PROTOCOL
DOI: 10.21769/BioProtoc.3796
Keywords
CRISPR/Cas9; Cas9-free; dual-sgRNA; gBlock; Genomic deletion
Categories
Funding
- Novo Nordisk Foundation [NNF15OC0014202]
- Copenhagen Plant Science Centre
- European Research Council (ERC) under the European Union [StG2017-757411]
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CRISPR/Cas9 system directed by a gene-specific single guide RNA (sgRNA) is an effective tool for genome editing such as deletions of few bases in coding genes. However, targeted deletion of larger regions generate loss-of-function alleles that offer a straightforward starting point for functional dissections of genomic loci. We present an easy-to-use strategy including a fast cloning dual-sgRNA vector linked to efficient isolation of heritable Cas9-free genomic deletions to rapidly and cost-effectively generate a targeted heritable genome deletion. This step-by-step protocol includes gRNA design, cloning strategy and mutation detection for Arabidopsis and may be adapted for other plant species.
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