CRISPR/Cas9-targeted mutagenesis in Caenorhabditis elegans.

The generation of genetic mutants in Caenorhabditis elegans has long relied on the selection of mutations in large-scale screens. Directed mutagenesis of specific loci in the genome would greatly speed up analysis of gene function. Here, we adapt the CRISPR/Cas9 system to generate mutations at specific sites in the C. elegans genome.

CURRENT methods to generate mutations in the genome of Caenorhabditis elegans, including chemical mutagenesis and imprecise excision of transposons, all rely on recovering mutations in large-scale mutagenesis screens. Recently, several groups reported the use of the Streptococcus pyogenes CRISPR/Cas9 system to generate double-strand break (DSB)-induced mutations in specific genomic loci in model systems including yeast (Dicarlo ), flies (Bassett ; Gratz ; Yu ), mammalian cells (Cho ; Mali ), and zebrafish (Hwang ). Because of the enormous potential for targeted genome engineering, we here investigate the suitability of the CRISPR/Cas9 system for use in C. elegans. This article is one of six companion articles in this issue (Chiu ; Cho ; Katic and Grosshans 2013; Lo ; Tzur ) that present different approaches to and features of Cas9-CRISPR genome editing in C. elegans.

The S. pyogenes CRISPR/Cas system effects site-specific cleavage of double-stranded DNA through a complex containing the Cas9 endonuclease and two noncoding RNAs (CRISPR RNA or crRNA, and trans-activating crRNA or tracrRNA) (Gasiunas ; Jinek ). Target site specificity is mediated by a 20-nt spacer region in the crRNA that is complementary to the target DNA and a 3-nt motif (NGG) following the target site in the DNA [termed protospacer adjacent motif (PAM)] (Gasiunas ; Jinek ). Thus a wide range of target sites can be chosen. Conveniently, a single synthetic guide RNA (sgRNA) that fuses the 3′ end of crRNA to the 5′end of tracrRNA is sufficient to target Cas9 to a specific site and generate DSBs (Jinek ) (Figure 1A).

Figure 1

Experimental design and germline Cas9 expression. (A) Cas9/sgRNA in complex with a target site. RuvC and HNH endonuclease domains together generate a double-strand break. In the sgRNA sequence, green bases are crRNA derived and red bases tracrRNA derived. (B) Schematic of the Cas9 expression vectors used in this study, placing Cas9 or Cas9::EGFP under control of the eft-3 promoter or the hsp-16.48 heat-shock promoter. Versions lacking EGFP are not shown. (C) Germline expression and nuclear localization of Cas9::EGFP expressed from the hsp-16.48 heat-shock promoter. Shown is a maximum-intensity projection of a Z-stack. Bar, 10 μm. (D) Diagrams and sequences of the U6::sgRNA and T7::sgRNA vectors. Gray background, promoter or downstream regions; orange background, sgRNA sequence downstream of the target recognition sequence; red background, BsaI recognition sites; boxed nucleotides, sequences left as 5′ overhang after BsaI digestion. (E) Example of cloning a target sequence into the U6::sgRNA vector. The 20-bp target site is outlined in blue and the PAM in yellow. The consensus sequences we used for target site selection are also indicated. Detailed materials and methods are available in File S1.

To promote expression of Cas9, we codon optimized the S. pyogenes Cas9 coding sequence for C. elegans, introduced artificial introns, and attached SV40 and nuclear localization signals to the N and C termini, respectively, of the encoded Cas9 protein (Figure 1B). To express Cas9 in the germline, we placed the Cas9 coding sequence under control of the or promoters and the 3′-UTR, each of which has been shown to be compatible with germline expression (Bessereau ; Merritt ; Frøkjær-Jensen ). To visualize expression of Cas9, we also generated Cas9::EGFP fusion vectors. We did not detect EGFP expression after injection of Peft-3::Cas9::EGFP (>20 animals examined). Injection of Phsp-16.48::Cas9::EGFP did result in visible EGFP expression, 5 hr after heat-shock induction for 1 hr at 34°. Expression did vary between experiments: one series of injections resulted in high expression in 5/5 animals examined (Figure 1C), while a second series of injections showed only weak expression in 1/12 animals examined. Because even low expression levels may provide sufficient enzymatic activity, in further experiments we tested both Peft-3- and Phsp-16.48-containing constructs for activity.

To provide the sgRNA, we tested two different approaches. First, we generated a vector containing a T7 promoter upstream of the sgRNA sequence for in vitro transcription of the sgRNA. Second, we generated a vector expressing the sgRNA under control of the regulatory sequences of an RNA polymerase III transcribed U6 snRNA on chromosome III, to enable in vivo transcription (Thomas ). Both vectors contain BsaI restriction sites for simple insertion of the target recognition sequence as an oligomer linker (Figure 1, D and E).

As a first test of functional activity, we generated a reporter construct carrying an out-of-frame copy of EGFP and lacZ downstream of the promoter. Imprecise repair of a DSB in a linker region between the first ATG and EGFP can result in a frameshift, leading to EGFP expression. We co-injected the reporter (15 ng/µl) with Peft-3::Cas9 or Phsp-16.48::Cas9 (50 ng/µl), a U6-driven sgRNA targeting the linker region (50 ng/µl), and a Pmyo-3::mCherry co-injection marker (5 ng/µl). We also tested injection of lower Cas9/sgRNA concentrations (20 ng/µl both) together with PstI-digested λ DNA (20 ng/µl), to promote generation of more complex extrachromosomal arrays. Per condition we injected 10 animals, and Phsp expression was induced by a 1-hr heat shock at 34° after the injection. None of the injections with Peft-3::Cas9 yielded viable transgenic F1’s. Instead, we observed mCherry-expressing dead embryos, indicating a deleterious effect of this construct. A series of test injections showed that the embryonic lethality is concentration dependent, ranging from 30% at 1 ng/µl to 100% at 20 ng/µl (see Supporting Information, Table S1). In contrast, 89% of the transgenic lines obtained from the injections with Phsp-16.48::Cas9 expressed EGFP in the pharynx, indicating the presence of an extrachromosomal array with at least one frame-shifted copy of the reporter (Table 1). The injection of a lower concentration Phsp::Cas9 diluted with λ DNA resulted in a higher number of transgenic offspring, although the fraction expressing EGFP was similar (90% and 84%, respectively, Table 1). Control injections lacking the sgRNA did not show EGFP expression, demonstrating specific Cas9/sgRNA activity (50 transgenic F1’s examined).

Table 1

Number of transgenic and EGFP-expressing F1 animals obtained using Cas9/sgRNA directed against an EGFP frameshift reporter

			Results
sgRNA concentrationa	Phsp-16.48::Cas9 concentrationa	No. P0 injected	Transgenic F1	F1 expressing EGFP
20	20	10	126	114 (90%)
50	50	10	32	27 (84%)

All concentrations are in nanograms per microliter. Injections with 20 ng/µl Cas9/sgRNA are supplemented with 20 ng/µl of PstI-digested λ DNA. All injections include 5 ng/µl of the Pmyo-3::mCherry marker to identify transgenic animals and 15 ng/µl of the out-of-frame EGFP reporter.

We also examined 18 stable transgenic lines obtained from EGFP-expressing F1 animals. Of these, 15 expressed EGFP in most (>90%) of the F2 transgenic animals. The small fraction of EGFP-negative transgenics could be due to mosaic inheritance of the extrachromosomal reporter array. Since Cas9 expression is induced by heat shock only in the injected P0 animals, these findings may indicate that DSBs were generated in the germline of the P0. Taken together, Cas9/sgRNA appears to efficiently generate DSBs in our plasmid-based reporter.

We next wanted to determine whether Cas9/sgRNA can be used to generate heritable mutations at a specific genomic locus in C. elegans. For this purpose, we generated sgRNA constructs targeting the coding sequence near the known mutation. We injected Phsp::Cas9 together with either in vitro transcribed sgRNA or the U6::sgRNA plasmid, as well as the Pmyo-3::mCherry co-injection marker (Table 2). For each combination we injected 20 P0 animals, selected individual F1 animals expressing mCherry, and examined their F2 progeny for the presence of Lin-5 offspring. Animals injected with in vitro-produced sgRNA failed to produce mutants (Table 2). In contrast, injections with U6::sgRNA yielded a total of 10 F1 animals that produced approximately one-quarter Lin-5 offspring (Table 2). We confirmed the presence of mutations at the locus by sequence analysis, identifying several deletions and a 7-bp insertion (Figure 2). For each F1 line we sequenced two mutant F2 animals independently, and in each case both animals harbored exactly the same mutation, strongly suggesting that the mutations were inherited from the parent and were not generated de novo by somatic events. Two mutations could not be resolved: Sanger sequencing traces from both sides degrade into double peaks at the sgRNA target site. This can result from the presence of a repeated sequence, and we speculate that DSB repair resulted in the duplication of a short DNA sequence. Injections with the lower concentration of Cas9 and sgRNA expression plasmids coupled with λ DNA yielded higher numbers of transgenic F1 animals, but ultimately produced the same number of mutants (Table 2).

Table 2

Number of transgenic F1 and mutant F2 progeny produced using Cas9/sgRNA directed against genomic loci

sgRNA				Transgenic F1
Method/target	Concentrationa	Phsp-16.48::Cas9 concentrationa	No. P0 injected	No. selected	With mutant progeny
U6 × lin-5	20	20	20	92	5
U6 × lin-5	50	50	20	24	5
T7 × lin-5	10	50	20	29	0
T7 × lin-5	150	50	20	124	0
U6 × rol-1	20	20	40	144	1
U6 × rol-1	50	50	20	140	2
U6 × dpy-11	50	50	20	20	2
U6 × unc-119	50	50	20	41	2

All concentrations are in nanograms per microliter. Injections with 20 ng/µl Cas9/sgRNA are supplemented with 35 ng/µl of PstI-digested λ DNA. All injections include 5 ng/µl of the Pmyo-3::mCherry marker to identify transgenic animals.

Figure 2

Genomic mutations generated by Cas9/sgRNA. Mutations are shown relative to the wild-type sequences. Three mutations could not be resolved by sequencing and may correspond to insertion of a repeated sequence. Blue indicates sgRNA target site, and yellow is the PAM motif.

Finally, we targeted three additional loci—, , and —using Phsp-16.48::Cas9 and U6::sgRNA (Table 2). As for , we selected transgenic F1 animals and looked for the presence of visible mutants in the F2 generation. For and , we identified two transgenic F1’s each that segregated approximately one-quarter mutant progeny, from a total of 20 and 41 transgenic F1 animals selected, respectively (Table 2). Homozygous mutations in or were readily identified in all cases (Figure 2). For , from 284 transgenic F1’s, we observed three plates with only a single Rol F2 animal. Sequencing of these mutants did confirm the presence of mutations at the target site (Figure 2). It appears therefore that the phenotypes generated by our sgRNA are only partially penetrant. Together, these results confirm the ability of our approach to generate mutations at specific loci in the genome.

Here, we adapted the CRISPR/Cas9 system for use in C. elegans and demonstrate its ability to efficiently generate genomic mutations. For , , and , we obtained on average one mutant from every 5 or 6 P0 animals injected. For , the frequency was much lower (three mutants of 60 P0 injections), but the partial penetrance of the phenotype likely caused us to miss several mutations. The approach is not only efficient but also fast: cloning, mutant isolation, and sequencing of mutations can be completed in 10 days.

A recently published CRISPR/Cas9 method for C. elegans uses Peft-3 to drive Cas9 expression (Friedland ). However, we found that expression of Cas9 from the promoter causes embryonic lethality. This contrasting result may be due to differences in the exact Cas9 protein produced. While the reason for the observed lethality is unclear, use of the heat-shock promoter to provide a pulse of expression only in the injected animal circumvents this problem.

Five companion articles (Chiu ; Cho ; Katic and Grosshans 2013; Lo ; Tzur ) also report the successful application of CRISPR/Cas9 in C. elegans. These groups use various approaches to provide Cas9 and sgRNA, including injection of Cas9 RNA or protein and in vitro-produced sgRNA. Thus, although in our case heat-shock-induced Cas9 coupled with U6-driven sgRNA proved most efficient, it appears that the methodology to provide these two components can be highly flexible.

In addition to generating mutants, the DSBs produced by Cas9/sgRNA enable several other applications of genome engineering, including insertion of exogenous DNA through homologous recombination (Katic and Grosshans 2013; Tzur ), and are likely to become an important tool for C. elegans researchers.