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Methods. 2017 May 15;121-122:86-93. doi: 10.1016/j.ymeth.2017.03.023. Epub 2017 Apr 7.

Precision genome editing using CRISPR-Cas9 and linear repair templates in C. elegans.

Author information

1
Dept of Molecular Biology and Genetics, Johns Hopkins University, School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA. Electronic address: apaix1@jhmi.edu.
2
Dept of Molecular Biology and Genetics, Johns Hopkins University, School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA. Electronic address: afolkma1@jhu.edu.
3
Dept of Molecular Biology and Genetics, Johns Hopkins University, School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA.

Abstract

The ability to introduce targeted edits in the genome of model organisms is revolutionizing the field of genetics. State-of-the-art methods for precision genome editing use RNA-guided endonucleases to create double-strand breaks (DSBs) and DNA templates containing the edits to repair the DSBs. Following this strategy, we have developed a protocol to create precise edits in the C. elegans genome. The protocol takes advantage of two innovations to improve editing efficiency: direct injection of CRISPR-Cas9 ribonucleoprotein complexes and use of linear DNAs with short homology arms as repair templates. The protocol requires no cloning or selection, and can be used to generate base and gene-size edits in just 4days. Point mutations, insertions, deletions and gene replacements can all be created using the same experimental pipeline.

KEYWORDS:

C. elegans; CRISPR; Cas9 RNP; Gene conversion; Genome editing; Homology-dependent repair (HDR); Linear repair templates; Short homology

PMID:
28392263
DOI:
10.1016/j.ymeth.2017.03.023
[Indexed for MEDLINE]
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