INTRODUCTION: 

A major goal of genetic engineering is to develop methods to modify the genome of animals easily and efficiently. CRISPR/Cas9 technology has greatly accelerated progress in this field by providing a simple method to introduce double-strand breaks in the genome. If a DNA with homology to the break (homology arms) is introduced at the same time as Cas9, the cell’s DNA repair machinery will use that DNA to repair the break and copy the sequence between the homology arms into the genome (homology-dependent repair - HDR).Our lab has found that, in C. elegans, homology-dependent repair is remarkably efficient (~100% of breaks repaired by HDR) provided that the repair templates are LINEAR and have short (~35 bases) homology arms that directly flank the Cas9 cleavage site (Paix et al., 2014, 2015).Linear DNAs (ssODNs and PCR amplicons) with 35-base homology arms are sufficient to introduce base- and gene-sized edits (up to 1.6kb).On the basis of these findings, we have developed a cloning-free protocol for editing the C. elegans genome. Our protocol does not require selection markers and uses the same experimental approach to mutate, tag, or delete any gene in the genome.

PROTOCOL OVERVIEW:

Requirements:

  1. A Cas9 recognition site(s) within 20 bases of the edit.
  2. An edit that will fit into a template <2kb long.

 

 

Experimental outline:

Day 0: Order gene-specific reagents:

    - crRNA(s) for Cas9 (can also be made in house)
    - ssODNs to use directly as template or to amplify template

You will also need Cas9 protein and tracr RNA.  Both are available commercially or can be made in house. See below for protocols to make your own Cas9 protein and crRNA.

 

Day 1: Assemble injection mix and inject 20 hermaphrodites

Injection mix contains:

    - Cas9 complex + template against gene of interest
    - Cas9 complex + template against dpy-10
dpy-10 is used to identify broods with the highest edit frequencies (co-conversion method: Arribere et al., 2014 Paix et al., 2015).   

 

Day 4 or 5: Identify 3 broods with highest numbers of dpy-10 edits (rollers). Clone out 48 Rollers from those broods.

 

Day 7 or 8: PCR genotype each F1 cohort.

 

Day 9: Clone out F2s to isolate homozygotes. 

 

Full editing protocol

PDF icon Full editing protocol.pdf

 

CAS9 protein purification
PDF icon Cas9 Protocol Purification SeydouxLab.pdf

 

Making crRNA RNA in house protocol

 

Other Protocols:

You should consider the following selection-based protocols if:

            - you prefer to use selection rather than PCR to identify your edits

            - there is no good Cas9 recognition sites within 20 bases of your edit

           - your edit involves an insert >2kb:

 

SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans.
Schwartz ML, Jorgensen EM.
Genetics. 2016 Feb 2. pii: genetics.115.184275. [Epub ahead of print]

 

Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette.
Dickinson DJ, Pani AM, Heppert JK, Higgins CD, Goldstein B.
Genetics. 2015 Aug;200(4):1035-49. doi: 10.1534/genetics.115.178335. Epub 2015 Jun 3.

 

Creating Genome Modifications in C. elegans Using the CRISPR/Cas9 System.
Calarco JA, Friedland AE.
Methods Mol Biol. 2015;1327:59-74. doi: 10.1007/978-1-4939-2842-2_6.

 

A recent review of CRISPR methods in C. elegans:

CRISPR-Based Methods for Caenorhabditis elegans Genome Engineering.
Dickinson DJ, Goldstein B.
Genetics. 2016 Mar;202(3):885-901. doi: 10.1534/genetics.115.182162.