CRISPR/Cas9

Yongquan Shen¹ and Ronald E Ellis¹

1 Department of Molecular Biology, Rowan University-SOM, Stratford, NJ 08084

Correspondence to: Ronald E Ellis PhD (ellisre@rowan.edu)

Genome editing with CRISPR/Cas9 or TALENs has become an essential tool for working with nematodes [3,4,5]. Plasmids expressing Cas9 and a custom sgRNA can be used for gene editing in many situations, particularly for C. elegans [2]. However, this approach is less effective for C.briggase and some male-female species. Furthermore, some edits will be difficult if a good Cas9 target is not located nearby.Our approach is to use Cas9 protein complexes for injections [4] when Cas9 target sites are available, and TALENs for other situations [5]. The best targets for Cas9 end in a 3’-GGnGG sequence [3], which is infrequent in worms. Although satisfactory sites can usually be found for gene knockouts, they might not be available for site-specific editing.

If possible, we use the native CRISPR/Cas9 system, which relies on Cas9 protein with both a universal RNA (tracrRNA) and a specific guide RNA (crRNA). These RNAs are bought from Dharmacon, and the same tracrRNA can be used for every injection. Although commercial Cas9 is also available, it is expensive. Thus, Paix and Seydoux developed a protocol for Cas9 production in E. coli [4]. We modified their procedure to simplify the Cas9 purification. Briefly, the plasmid NM2973 was transformed into NEB Nico-21 competent cells (#C2529H), and Cas9 expression was induced by adding IPTG to the culture media. We purified Cas9 under native conditions, using a Qiagen Fast-Start Kit (#30600). Following instructions, the bacterial pellet was resuspended in 10 ml of lysis buffer, and cooled on ice for 30 minutes. The cell lysate supernatant flowed through the Fast-Start Ni-NTA column by gravity, and 4 ml of washing buffer was used to wash the column twice. Next, Cas9 was eluted with 2 ml of elution buffer. Finally, the Cas9 eluate was centrifuged with a centricon filter (#UFC905024) to concentrate the protein. We obtained 2.5 mg of Cas9 from 1L of culture, and made 2 µl aliquots at a concentration of 10 µg/µl for storage at -80°C.

Home-made Cas9 was tested to measure it efficiency. First, we performed a cleavage assay to confirm that it had endonuclease activity. A mixture of Cas9, mouse SLC guide RNA and a PCR fragment from the SLC gene were digested at 37°C for 1 hour. The result showed that our Cas9 completely cleaved the SLC fragment as shown in Figure 1. Second, we used the Cas9 to knockout the cbr-dpy-5 gene in vivo. The synthetic cbr-dpy-5 crRNA and tracrRNA were incubated with Cas9 at 37°C for 15 minutes, and microinjected into the gonads of C. briggase hermaphrodites. Among all the F1 progeny, 25% produced homozygous Dpy children. A similar injection using commercial Cas9 (PNA Bio.) gave 18% affected F1s. Sequencing confirmed that the cbr-dpy-5 mutation was a 51 bp deletion near the PAM site of its target. Thus, home-made Cas9 has high efficiency and allows rapid and inexpensive gene editing.

If the desired target lacks a nearby 3’-GGnGG, TALENs can be used for precise gene editing. Since the only constraint for TALEN binding is that the first nucleotide in each target sequence is a T, sites exist almost everywhere in the genome. The plasmids to synthesize a pair of TALEN mRNAs can easily be built in a week, using a procedure developed by Cermak et al [1], with the destination vector pRE189 for expressing nematode mRNAs [5]. A Golden Gate TALEN kit is available from Addgene (#1000000024) and mRNA kits from Ambion (#AM1344, #AM1908).

Acknowledgments: We thank Dr. Geraldine Seydoux for the NM2973 plasmid. This work was supported by NIH grant to Dr. Ronald E Ellis.

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Figure 1: Cas9 Cleavage assay in vitro

References

[1] Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, and Voytas DF. (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39(12):1-11

[2] Dickinson DJ, And Goldstein B. (2016) CRISPR-based methods for Caenorhabditis elegans genome engineering. Genetics. 202(3):885-901

[3] Farbound B, and MeyerBJ. (2015) Dramatic enhancement of genome editing by CRISPR-Cas9 through improved guide RNA design. Genetics. 199(4):959-971

[4] Paix A, Kolkmann A, Rasoloson D, and Seydoux G. (2015) High efficiency, homology-directed genome editing in Caenorhabditis elegans using CRISPR-Cas9 ribonucleoprotein complexes. Genetics. 201(1):47-54

[5] Wei Q, Shen Y, Chen X. Shifman Y. and Ellis RE. (2014) Rapid creation of forward genetics tools for C. briggase using TALENs: lessons from nonmodel organisms. Mol. Biol. Evol. 31(2):468-473

Alakananda Das¹, Daniel Dickinson², Bob Goldstein² and Kevin Slep²

¹Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. ²
Department of Biology, University of North Carolina at Chapel Hill,
Chapel Hill, NC, USA.

Correspondence to: Alakananda Das (alkadask@stanford.edu)

Cas9-targeted homologous recombination methods are now widely used for tagging C. elegans genes at their endogenous genetic loci. The primary advantage of this method is that it involves minimal modifications to the native regulatory elements controlling gene expression. This is preferable for experiments that require the spatio-temporal control of tagged protein expression to be comparable to native expression. However, it is often desirable to study a protein of interest in the context of a particular cell or tissue that represents a subset of its native expression profile in the worm. For genes that are expressed in a wide range of cell types, signal from tagged protein expression from neighboring cells may mask the signal from the particular cell or tissue under investigation. Recently, Schwartz et al. described a FLP/FRT recombination based method to append cell/tissue specific tags at the N- or C terminus of C. elegans genes (Schwartz et al., 2016). Here we describe an alternative method using the Cre-Lox recombination system to selectively append tags at the endogenous genetic locus only in cells or tissues of interest, while ensuring untagged protein expression in other cells.

The C. elegans che-12 gene is expressed in a subset of ciliated sensory neurons in the head and tail of the animal, including the amphid neurons with simple rod like cilia (Bacaj et al., 2008). CHE-12 localizes to the cilia in these neurons which form a bundle in the amphid channel. Previously, we observed CHE-12::GFP puncta along the amphid cilia suggesting coupling to intraflagellar transport machinery, however, the arrangement of amphid cilia in a bundle inside the amphid channel presented a challenge to follow a single fluorescent particle continuously from the cilia base to the distal tip (Das et al., 2015). In order to better visualize CHE-12 dynamics in a single amphid cilium, we devised a strategy to fluorescently tag the endogenous che-12 gene in a single amphid neuron, the ASER neuron.

Our cell-specific tagging cassette (CTC) includes a Stop codon and a strong terminator sequence (let-858 3’UTR) bounded at either end by two LoxP sites within short synthetic introns, followed by the coding sequence of any fluorescent protein (Figure 1A). We inserted a hygromycin resistance gene in the inter-genic region between the che-12 and B0024.4 genes for the selection of positive knock-in candidates. The CTC is introduced between the end of the coding sequence of the gene of interest and its stop codon using Cas9-targeted homologous recombination methods described in Dickinson et al., 2013. Animals bearing a correct insertion of the CTC at the selected genetic locus express the untagged version of the protein. A cell/tissue specific promoter is then used to drive the expression of Cre-recombinase in the specific cell/tissue where expression of the fluorescently tagged endogenous protein is desired. Cre-recombinase will excise out the region flanked by the LoxP sites in these specific cells, creating a fusion of the gene of interest and the fluorescent protein coding sequence, leading to cell/tissue-specific tagging of endogenous proteins. See Figure 1A an illustration of this strategy.

We introduced a CTC as outlined in Figure 1A at the 3’-end of the che-12 coding sequence using Cas9-targeted homologous recombination. We then used a ubiquitous or cell/tissue-specific promoter-driven Cre-recombinase, expressed from an extra-chromosomal array to allow selective recombination in those specific cells and fuse the che-12 coding sequence with mNeonGreen coding sequence. This strategy was successful in selectively labeling CHE-12 in a single neuron when we used gcy-5 promoter-driven Cre-recombinase (Figure 1B). In contrast, when we used a ubiquitous promoter such as Peft-3 to drive Cre-recombinase expression, we observed CHE-12::mNeonGreen signal in all amphid neurons (Figure 1C). The tagged CHE-12::mNeonGreen fusion protein localized correctly to the ASER cilium (Figure 1B and 1D).

This cell-specific tagging strategy is applicable for cell and tissue types for which strong cell/tissue-specific promoters have been characterized. This strategy can also be modified to delete domains within a protein or the entire coding sequence of a gene in a desired cell type by introducing synthetic introns containing embedded LoxP sites at the boundaries of a segment targeted for deletion.

Figures

References

Bacaj T, Lu Y, and Shaham S. (2008). The conserved proteins CHE-12 and DYF-11 are required for sensory cilium function in Caenorhabditis elegans. Genetics. 178, 989-1002.

Das A, Dickinson DJ, Wood CC, Goldstein B, and Slep KC. (2015). Crescerin uses a TOG domain array to regulate microtubules in the primary cilium. Mol Biol Cell. 26, 4248-4264.

Dickinson DJ, Ward JD, Reiner DJ, and Goldstein B. (2013). Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat Methods. 10,1028-1034.

The WBG

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Setting up simple and cost-effective gene editing for nematodes

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References

Cell-specific tagging of C. elegans genes using Cas9-targeted homologous recombination

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References