Homology-directed repair (HDR) of breaks induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing in several organisms. Most HDR protocols rely on plasmid-based expression of Cas9 and the gene-specific guide RNAs. Here we report that direct injection of in vitro–assembled Cas9-CRISPR RNA (crRNA) trans-activating crRNA (tracrRNA) ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high frequency. Building on our earlier finding that PCR fragments with 35-base homology are efficient repair templates, we developed an entirely cloning-free protocol for the generation of seamless HDR edits without selection. Combined with the co-CRISPR method, this protocol is sufficiently robust for use with low-efficiency guide RNAs and to generate complex edits, including ORF replacement and simultaneous tagging of two genes with fluorescent proteins.
THE CRISPR-Cas9 system is a bacterial adaptive immune system that has been harnessed as a powerful genome editing tool (Doudna and Charpentier 2014). Cas9 is a nuclease that functions with two small RNAs: CRISPR RNA (crRNA), which guides Cas9 to complementary target sequences, and trans-activating crRNA (tracrRNA), which binds to the crRNA and to Cas9 to form the ribonucleoprotein (RNP) complex (Deltcheva et al. 2011). For use in genome editing, the crRNA and tracrRNA are often combined into a single chimeric guide RNA (sgRNA) (Jinek et al. 2012). Expression of Cas9 and sgRNA in cells leads to cleavage of complementary genomic sequences. The double-strand breaks are repaired by endogenous cellular pathways, including end-joining mechanisms [e.g., nonhomologous end joining (NHEJ) and theta-mediated end joining (TMEJ)] and homology-dependent repair (HDR) mechanisms (van Schendel et al. 2015). End joining typically introduces random insertions/deletions at the DNA break site, which can disrupt gene activity. HDR, in contrast, is a more precise repair process that uses a repair template. If the repair template contains edits flanked by sequences that are homologous to the cleavage site (homology arms), the edits will be incorporated by gene conversion. The high efficiency of Cas9 and the simplicity of guide RNA design have made it possible to develop end-joining protocols to systematically knock out genes (Hsu et al. 2014; Shah et al. 2015). In principle, scalable HDR protocols also could be used for systematic knock-ins of custom edits [such as green fluorescent protein (GFP)]. Unfortunately, current HDR protocols are inefficient (Hsu et al. 2014). First, most HDR protocols require cloning to create a repair template and a guide RNA expression vector for each gene to be targeted. Second, the efficiency of HDR typically is low, requiring the screening of large number of animals or the use of selection markers that are integrated alongside the desired edits (Dickinson et al. 2013, 2015).
Recently, we found in Caenorhabditis elegans that linear DNAs with homology arms as short as 35 bases can support the efficient incorporation of HDR edits (Paix et al. 2014). This finding simplifies the construction of donor templates. For example, a donor template to introduce GFP can be synthesized using two 55-base oligos, each containing 35 bases that are homologous to the targeted locus and 20 bases that are homologous to GFP. The PCR amplicon is injected alongside plasmids coding for Cas9 and the sgRNA into the gonad of adult hermaphrodites, and their progeny are screened for GFP expression by visual inspection or by PCR screening. Using this method, we were able to recover GFP fusions at six of eight loci attempted. HDR frequencies were high enough to avoid the use of coselection makers but were still relatively low and variable (0.4–12%) (Paix et al. 2014). Other researchers also have reported difficulties in obtaining HDR edits, a problem that can be partially overcome by improvements in the design of guide RNAs and repair templates (Farboud and Meyer 2015; Katic et al. 2015).
Two additional factors are likely to also contribute to low edit frequency. First, the efficiency of expressing and assembling Cas9–sgRNA complexes from plasmids is not known and may be low given the tendency of C. elegans germ cells to silence foreign DNA (Kelly et al. 1997). In mouse embryos and mammalian cells, direct delivery of Cas9 complexes assembled in vitro has been reported to yield a higher frequency of edits (Lin et al. 2014; Aida et al. 2015; Liang et al. 2015). Injection of Cas9 complexes has been used in C. elegans to create edits by end-joining mechanisms (Cho et al. 2013) but has not yet been compared directly to plasmid delivery or used for HDR.
A second factor that contributes to the low edit frequency is that many injected hermaphrodites generate no edits. For reasons that remain unclear, we have found that edits tend to cluster among the progeny of a minority of injected mothers (jackpot broods) (Paix et al. 2014). Identification of jackpot broods before screening would eliminate the need to screen the broods of nonproductive hermaphrodites. Recently, three reports have described methods to enrich for desired edits by selecting or screening for editing at a second marker locus (Arribere et al. 2014; Kim et al. 2014; Ward 2015). In the method described by Arribere et al. (2014), a dominant mutation is introduced by HDR in the marker locus dpy-10, leading to a roller phenotype that is easily identified among the progeny of injected hermaphrodites.
Here we report the development of a new direct-delivery protocol that combines injection of in vitro–synthesized and –assembled Cas9-crRNA-tracrRNA complexes with the dpy-10 co-CRISPR approach of Arribere et al. (2014) to identify jackpot broods. The direct-delivery protocol is entirely cloning-free and generates edits at frequencies ranging from 2 to 70% of F1 progeny, a 10-fold improvement over our plasmid-based earlier method (Paix et al. 2014). This new protocol permits the use of inefficient sgRNAs that failed in our previous study and expands the repertoire of possible genome edits to include ORF swaps and fluorescent protein (FP) tagging of two genes at once.
Materials and Methods
Supplementary materials include protocols for direct-delivery editing (Supporting Information, File S1) and for Cas9 protein purification (File S2), as well as lists of plasmids (Table S2), crRNAs (Table S3), rescue templates (Table S4), and strains (Table S5).
Strains and plasmids are available from the Caenorhabditis Genetics Center (CGC) and Addgene, or upon request.
Results and Discussion
Injection of Cas9 RNP complexes supports robust HDR
We developed a simple method to purify from Escherichia coli recombinant Cas9 [fused at its C-terminus with a nuclear localization sequence (Fu et al. 2014)] and assemble Cas9-crRNA-tracrRNA RNP complexes in the presence of repair templates and in a buffer suited for injection into C. elegans (File S1 and File S2). Cas9 also can be obtained from a commercial source, as in Cho et al. (2013). As in the co-CRISPR method of Arribere et al. (2014), we co-injected Cas9 complexes and repair templates targeting the marker gene dpy-10 and a second target locus. dpy-10 was repaired with a single-strand oligodeoxyribonucleotide (ssODN) that introduces a missense mutation in the dpy-10 ORF (Arribere et al. 2014). The missense mutation causes a dominant roller phenotype that is easily spotted under a dissecting microscope. For the second locus, we first used gtbp-1 (aka K08F4.2), a nonessential gene expressed in most tissues, which we targeted previously using plasmid delivery (Paix et al. 2014). We introduced a single cut near the C-terminus of gtbp-1 and repaired the lesion with an ∼700-base double-stranded PCR fragment containing the ORF for a fluorescent protein [red fluorescent protein (RFP) or GFP] flanked by 35-base arms that were homologous to sequences immediately surrounding the cut site (Figure 1).
For three experiments (Table 1 and Table S1, experiments AP58, AP60, and AP78), we examined the broods of all injected hermaphrodites by visual inspection for the presence of roller and fluorescent (FP+) worms (Figure 2). We found that 50–61% of injected hermaphrodites segregated rollers (Table 1). Broods without rollers contained no FP+ edits (experiment AP58: 0 of 62 F1 progeny examined from five broods). Among broods with rollers, edits could be found in both rollers and their nonroller siblings (Table 1). For each experiment, we found that the frequency of FP+ edits among the three broods that segregated the highest number of rollers was higher than that observed among the three broods with the lowest numbers of rollers (Figure 2), as also reported by Arribere et al. (2014). For simplicity, in subsequent experiments, we analyzed only the three broods with the highest number of rollers (jackpot broods) and ignored all other broods. We conclude that direct delivery of Cas9 RNP complexes supports robust HDR and, like plasmid delivery, generates jackpot broods that are easily identified using the dpy-10 co-CRISPR marker (Arribere et al. 2014).
Increasing the target/marker ratio of crRNAs and repair templates increases HDR frequency at the target locus
When using equimolar amounts of crRNA and templates for dpy-10 and gtbp-1 (Table 1, experiment AP80), we obtained FP+ edits at a higher frequency among rollers than among nonrollers (24 vs. 3%) (Table 1). For genes unlinked to dpy-10, edits of interest can be separated from the dominant dpy-10(Rol) edit by picking nonroller worms in the next generation. For genes linked to dpy-10, however, recovering the edit without the marker is more difficult, and it is therefore preferable to isolate the edit in nonroller F1 progeny. We reasoned that increasing the levels of gtbp-1 crRNA and template relative to that of dpy-10 should increase the frequency of gtbp-1 edits overall, including in the nonroller population. As expected, increasing the gtbp-1/dpy-10 ratio of crRNA (experiments AP80 and AP78) and template (experiments AP78 and AP96) increased the number of GFP+ edits (Table 1 and Table S1). We also found, however, that decreasing the concentration of dpy-10 crRNA below 5 pmol/µl made it difficult to obtain enough rollers for analysis (experiment AP108). Similarly, the need to maintain relatively high overall concentrations of Cas9 complexes limits the amount of excess target template that can be added to the injection mix (see protocol in File S1 and Table 1, experiments AP78 and AP111). Under the best conditions tested (experiment AP58, high gtbp-1/dpy-10 ratio for both the crRNAs and the repair templates), 61% of injected hermaphrodites segregated rollers, and the jackpot broods gave rise to 61% FP+ edits among rollers and 52% FP+ edits among nonrollers.
Direct injection of preassembled Cas9 RNP complexes generates more overall edits per injected hermaphrodite than plasmid delivery
The FP+ edit frequencies shown in Table 1 are four- to fivefold higher than those obtained using plasmid delivery at the same locus without co-CRISPR (12%) (Paix et al. 2014) or at a different locus using co-CRISPR and longer homology arms (14%) (Arribere et al. 2014). To directly compare the efficiency of direct delivery vs. plasmid delivery, we injected plasmids expressing Cas9 and sgRNAs targeting gtbp-1 and dpy-10 with the same high gtbp-1/dpy-10 ratio as in experiment AP78. Of 17 injected hermaphrodites, we obtained only two (11%) that segregated rollers, for a total of 18 rollers (Table 1, experiment AP112). This low roller frequency is consistent with that reported by Arribere et al. (2014). The frequency of FP+ worms across the two roller broods, however, was high (83% among rollers, 14% among nonrollers). These findings confirm that plasmid delivery can generate jackpot broods with high edit frequencies. Direct delivery of preassembled Cas9 RNP complexes, however, yields a higher number of productive injections, i.e., injected hermaphrodites that generate rollers (50 vs. 11%). In practice, this means that fewer hermaphrodites (∼10 instead of ∼50) need to be injected to obtain jackpot broods.
Application of the protocol to challenging edits
We reasoned that the higher efficiency of our protocol might make it possible to use low-efficiency sgRNAs. Previously, using plasmid delivery and no co-CRISPR, we failed to recover GFP edits at two loci, glh-1 and htp-3, likely because the sgRNAs targeting those genes failed to support sufficient cutting to stimulate HDR (Paix et al. 2014). Both of these sgRNAs have a C at their 3′ ends, which is thought to be unfavorable (Doench et al. 2014). We retested the same sgRNAs and repair templates using complexes assembled in vitro at high target/dpy-10 crRNA and template ratios. We obtained 47% edits for glh-1 and 2% edits for htp-3 (Table 2 and Table S1, experiments AP106 and AP114). A recent report has shown that guide RNAs that end in GG consistently yield good editing efficiencies in C. elegans (Farboud and Meyer 2015). We tested such a sgRNA at the gtbp-1 locus and obtained 20% edits (Table 2, experiment AP92), a frequency comparable to that of the original sgRNA we used at that locus, which ends in a single G. We conclude that the higher efficiency of the new protocol expands the range of sgRNAs that can be used, although efficiencies still vary greatly (Table S3).
Insertion at a distance from Cas9-induced cleavage site:
We previously noted that edits at a distance from the cleavage site (>10 bases) are incorporated less efficiently than edits closer to the cleavage site (Paix et al. 2014). We found that this observation still holds using the new protocol. We obtained 6% edits when inserting GFP in gtbp-1 28 bases away from the cleavage site (Table 2, experiment AP83) compared to 51% edits when inserted directly at the cleavage site under the same crRNA and template ratios (Table 1, experiment AP78). A low edit frequency also was observed when using a ssODN to insert a small tag at the same position away from the cleavage site (Table 2, experiment AP82). Increasing the ssODN concentration improved efficiency modestly (experiment AP95, 2 vs. 8.8%). We conclude that edits away from the cleavage site are incorporated less efficiently than edits close to the cleavage site, as reported previously (Arribere et al. 2014; Paix et al. 2014). This inefficiency can be compensated partially by increasing the template concentration in the injection mix.
Tagging two genes at once:
We tested whether the new protocol could be used to recover edits at two target loci simultaneously (in addition to the marker locus dpy-10). We targeted gtbp-1 and fbf-2 for GFP and RFP insertion, respectively. We obtained 28% GFP/RFP double edits among rollers and 7% GFP/RFP double edits among nonrollers (Table 2, experiment AP103). The frequency of GFP edits overall (63%) exceeded that of RFP edits (35%) almost by a factor of 2 (Table S1), possibly as a result of higher efficiency of the sgRNA targeting gtbp-1 compared to fbf-2, as also seen when these sgRNAs were used separately (Paix et al. 2014). We conclude that protein delivery can support robust HDR at three loci simultaneously (including dpy-10) even when using sgRNAs with different editing efficiencies.
We showed previously that a repair ssODN could be used to insert a restriction site at the junction of a deletion generated by two cuts. To test whether a similar approach could be used to replace an entire ORF with another, we first attempted to replace the gtbp-1 ORF with a histone H2B::GFP fusion. We recovered 58% replacements among rollers and 7.5% replacements among nonrollers (Table 2, experiment AP115). Next, we attempted to replace GFP with RFP at two loci previously tagged with GFP. We used a pair of crRNAs that cut at the 5′ and 3′ ends of GFP and an RFP PCR repair template with 35-base homology arms directly flanking the cleavage sites. We obtained the desired GFP-to-RFP swap in 26% (gtbp-1 locus) and 21% (deps-1 locus) of rollers. Interestingly, we also recovered a significant number of rollers that were GFP− and RFP−, indicating a high degree of cutting in the GFP target without HDR (Figure 3 and Table S1). Because both maternal and paternal GFP copies were affected, these observations indicate that Cas9 complexes injected in the maternal germline persist into zygotes, where they can edit the paternal genome, as also reported by Cho et al. (2013). Consistent with these observations, in addition to rollers, in all our experiments we also obtained dumpy worms, which result from editing both copies of the dpy-10 locus (Arribere et al. 2014).
We report that injection in the C. elegans gonad of repair templates and preassembled Cas9-crRNA-tracrRNA complexes generates HDR edits with high efficiency (∼50% of injected hermaphrodites generate edits). Direct delivery allows us to take full advantage of the benefits of the co-CRISPR approach of Arribere et al. (2014), which provides a visual marker to identify broods with edits. By optimizing the ratio of Cas9 complexes (and repair templates) that target the gene of interest compared to the dpy-10 marker, we found that it is possible to obtain both a high yield of dpy-10-edited progeny and a high yield of dpy-10 edits that are also edited at the gene of interest (coedits, as high as 70%). Among broods that segregate the highest numbers of dpy-10 edits (jackpot broods), edits at the gene of interest also can be found in non-dpy-10-edited siblings (as high as 52%). The ability to recover edits in unmarked animals is particularly useful for linked loci that cannot be separated easily from the dominant dpy-10 marker in the next generation. Our findings also demonstrate that the co-CRISPR strategy works efficiently even when using different types of templates to repair the marker locus (ssODN to generate a point mutation) and the gene of interest (PCR amplicon to insert a fluorescent protein). The efficiency of editing, however, still remains variable from locus to locus, likely owing to differences in sgRNA efficiency and possibly differences in locus competency for HDR. We also continue to find that edits at a distance from the Cas9 cleavage site (>10 bases) are incorporated less efficiently than edits closer to the cleavage site, as reported previously (Arribere et al. 2014; Paix et al. 2014). Although protospacer adjacent motifs (PAMs) can be found within 30 bases of most edit sites, in cases where no PAMs are available, it may be preferable to use templates with longer homology arms and selection markers, as in Dickinson et al. (2015).
The efficiency of our direct-delivery protocol translates into several practical advantages: workload is reduced at both the injection (10–20 injected hermaphrodites vs. 50) and screening (<50 F1 progeny compared to hundreds) steps, low-efficiency sgRNAs can be used, and complex edits, including gene replacements and multigene edits, are possible. Replacement of an ORF with GFP, as we report here for the gtbp-1 locus, is an effective method to generate marked null alleles that can be maintained using the linked fluorescent marker. We have also developed universal crRNAs and rescue templates to convert GFP fusions to RFP fusions (File S1). Another advantage of direct delivery is that the entire protocol requires no cloning: tracrRNA, crRNAs, and ssODN repair templates for short edits (<50 bases) are all synthesized chemically, and longer repair templates are made by PCR using oligonucleotides to code for the short (35-base) homology arms. In principle, direct delivery of Cas9 RNP complexes could be combined with other CRISPR protocols (Dickinson et al. 2015; Ward 2015) and also should be advantageous for nematodes where promoters to drive expression of Cas9 and the sgRNAs are not readily available (Chiu et al. 2013; Witte et al. 2015).
We thank Mike Nonet and Andy Fire for the Cas9::NLS::His bacterial expression plasmid and members of the Seydoux Laboratory for comments on the manuscript. Some strains were provided by or deposited at the Caenorhabditis Genetics Center (CGC), which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440). We also thank WormBase for gene annotations and sequences. This work was supported by National Institutes of Health grant R01HD37047. G.S. is an investigator of the Howard Hughes Medical Institute.
Communicating editor: O. Hobert
Supporting information is available online at www.genetics.org/lookup/suppl/doi:10.1534/genetics.115.179382/-/DC1
- Received June 12, 2015.
- Accepted July 9, 2015.
- Copyright © 2015 by the Genetics Society of America
Available freely online through the author-supported open access option.