CRISPR/Cas9 mediated gene editing in non-model nematode Panagrolaimus sp. PS1159
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Abstract
The phylum Nematoda harbors a huge diversity of species in a broad range of ecosystems and habitats. Nematodes share a largely conserved Bauplan but major differences have been found in early developmental processes. The development of the nematode model organism Caenorhabditis elegans has been studied in great detail for decades. These efforts have provided the community with a large number of protocols and methods. Unfortunately, many of these tools are not easily applicable in non- Caenorhabditis nematodes. In recent years it has become clear that many crucial genes in the C. elegans developmental toolkit are absent in other nematode species. It is thus necessary to study the developmental program of other nematode species in detail to understand evolutionary conservation and novelty in the phylum. Panagrolaimus sp. PS1159 is a non-parasitic nematode exhibiting parthenogenetic reproduction and we are establishing the species to comparatively study evolution, biodiversity, and alternative reproduction and survival strategies. Here, we demonstrate the first successful application of the CRISPR/Cas9 system for genome editing in Panagrolaimus sp. PS1159 and the closely related hermaphroditic species Propanagrolaimus sp. JU765 applying the non-homologous end joining and the homology-directed repair (HDR) mechanisms. Using microinjections and modifying published protocols from C. elegans and P. pacificus we induced mutations in the orthologue of unc-22. This resulted in a visible uncoordinated twitching phenotype. We also compared the HDR efficiency following the delivery of different single-stranded oligodeoxynucleotides (ssODNs). Our work will expand the applicability for a wide range of non-model nematodes from across the tree and facilitate functional analysis into the evolution of parthenogenesis, changes in the developmental program of Nematoda, and cryptobiosis.
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After 12 days (generation time is 8-10 days in Panagrolaimus; Schiffer et al., 2019), 16 F1 animals showing reduced crawling activity and a twitching phenotype in water were transferred into single drops of Plectus nematode growth medium (PNGG; supplementary Table S1)
It would be helpful to explicitly state the values shared in Table 4 within the text to provide readers some context about the efficiency of recovering a phenotype.
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A cladogram showing the position of the two main species in this study, Panagrolaimus sp. PS1159 and Propanagrolaimus sp. JU765, in relation to the nematode model organisms C. elegans and P. pacificus, as well as A. rhodensis and S. ratti, species with reportedly successful use of the CRISPR system
It might be nice to indicate which species have the features/ methods of interest already developed. For example, you could have a number of columns to the right of the cladogram with a colored circle for:
a) specific biological features, such as life history (parthenogenesis vs. hermaphrodism); b) whether or not RNAi methods have been demonstrated; c) whether or not CRISPR/Cas9 methods have been demonstrated.
This could be a nice way to put the findings in this manuscript into a broader context with a figure.
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To prepare injection needles using a puller (Sutter Instruments, P-2000), we use a 1-lined so called “bee stinger” program (Oesterle, 2018) as a basis for a 3-lined program (table 1), producing sharper needles with a short taper.
If available, it might be useful for others familiar with nematode injection if an image of this needle compared to a standard needle could be included in the supplement.
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During the first experiments, we were only able to isolate a few worms with a twitching phenotype and injection results were thus unsatisfactory and not consistent.
Would the authors be able to place the efficiency in the context of existing literature for C. elegans or P. pacificus, for example?
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From these results we decided that the preferred time window to screen for mutations after injections, is 48 hours post injection after considering the feasibility and simplicity of maintenance of the progeny and achieving mutations arising from a higher number of P0s.
Would this optimal timing potentially vary depending on what the specific phenotype of interest might be?
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We also tested 1300 wild-type PS1159 for spontaneous twitching behavior in water and 1% nicotine for 10 minutes to analyze if this phenotype could frequently occur in a wild-type population, but could not observe any.
While the reported phenotypes (motility and survival) are convincing, if the authors performed such experiments, it might be helpful to know whether any sham injections (buffer only, Cas9 only, guide RNA only) are able to produce any offspring with the uncoordinated phenotype.
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In Pristionchus pacificus the CRISPR mix protocol differs from the one used in C. elegans (Paix et al., 2017) by an additional pre-annealing step of the tracrRNA and crRNA (James Lightfoot, pers. comment). We tested if the incubation temperature has an impact on the complex formation in PS1159. We therefore injected the complex with the modified crRNA with 60 µM concentration. When pre-annealing the crRNA and tracrRNA at 95 °C for 5 minutes (Hiraga et al., 2021) at the beginning we yielded 8.25% efficiency. When assembling the mix without the annealing step at 95 °C, but with an incubation step at 37 °C for 10 minutes after adding the Cas9 we reached to 11.57% of twitching progeny. When using both steps, the percentage of F1s with the phenotype increased up to 16.34 (Table 2).
The authors indicate in Table 2 that some experiments were …
In Pristionchus pacificus the CRISPR mix protocol differs from the one used in C. elegans (Paix et al., 2017) by an additional pre-annealing step of the tracrRNA and crRNA (James Lightfoot, pers. comment). We tested if the incubation temperature has an impact on the complex formation in PS1159. We therefore injected the complex with the modified crRNA with 60 µM concentration. When pre-annealing the crRNA and tracrRNA at 95 °C for 5 minutes (Hiraga et al., 2021) at the beginning we yielded 8.25% efficiency. When assembling the mix without the annealing step at 95 °C, but with an incubation step at 37 °C for 10 minutes after adding the Cas9 we reached to 11.57% of twitching progeny. When using both steps, the percentage of F1s with the phenotype increased up to 16.34 (Table 2).
The authors indicate in Table 2 that some experiments were summarized together. It seems that the specific comparison being made in this section could benefit from some additional repeated trials to see whether the differences in annealing temperatures reproducibly result in a difference in phenotype recovery.
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The authors of this study demonstrate CRISPR/Cas9 mutagenesis and knock-in for the non-model nematodes Panagrolaimus sp. PS1159 and Propanagrolaimus sp. JU765 using multiple orthogonal approaches, including behavioral analysis, chemical perturbation, nucleic acid biochemistry (T7, restriction digest), and Sanger sequencing. The results presented are well-supported and enable functional genetic perturbations in these organisms.
There are a few cases where additional data might strengthen the authors' claims, which I've marked. In some locations, the authors could provide a bit more context for their results for the ease of reader comprehension. I find that the explanation of the authors' more anecdotal observations is particularly helpful, especially for others who might try similar experiments in diverse nematodes.
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The authors of this study demonstrate CRISPR/Cas9 mutagenesis and knock-in for the non-model nematodes Panagrolaimus sp. PS1159 and Propanagrolaimus sp. JU765 using multiple orthogonal approaches, including behavioral analysis, chemical perturbation, nucleic acid biochemistry (T7, restriction digest), and Sanger sequencing. The results presented are well-supported and enable functional genetic perturbations in these organisms.
There are a few cases where additional data might strengthen the authors' claims, which I've marked. In some locations, the authors could provide a bit more context for their results for the ease of reader comprehension. I find that the explanation of the authors' more anecdotal observations is particularly helpful, especially for others who might try similar experiments in diverse nematodes.
-
From these results we decided that the preferred time window to screen for mutations after injections, is 48 hours post injection after considering the feasibility and simplicity of maintenance of the progeny and achieving mutations arising from a higher number of P0s.
Would this optimal timing potentially vary depending on what the specific phenotype of interest might be?
-
In Pristionchus pacificus the CRISPR mix protocol differs from the one used in C. elegans (Paix et al., 2017) by an additional pre-annealing step of the tracrRNA and crRNA (James Lightfoot, pers. comment). We tested if the incubation temperature has an impact on the complex formation in PS1159. We therefore injected the complex with the modified crRNA with 60 µM concentration. When pre-annealing the crRNA and tracrRNA at 95 °C for 5 minutes (Hiraga et al., 2021) at the beginning we yielded 8.25% efficiency. When assembling the mix without the annealing step at 95 °C, but with an incubation step at 37 °C for 10 minutes after adding the Cas9 we reached to 11.57% of twitching progeny. When using both steps, the percentage of F1s with the phenotype increased up to 16.34 (Table 2).
The authors indicate in Table 2 that some experiments were …
In Pristionchus pacificus the CRISPR mix protocol differs from the one used in C. elegans (Paix et al., 2017) by an additional pre-annealing step of the tracrRNA and crRNA (James Lightfoot, pers. comment). We tested if the incubation temperature has an impact on the complex formation in PS1159. We therefore injected the complex with the modified crRNA with 60 µM concentration. When pre-annealing the crRNA and tracrRNA at 95 °C for 5 minutes (Hiraga et al., 2021) at the beginning we yielded 8.25% efficiency. When assembling the mix without the annealing step at 95 °C, but with an incubation step at 37 °C for 10 minutes after adding the Cas9 we reached to 11.57% of twitching progeny. When using both steps, the percentage of F1s with the phenotype increased up to 16.34 (Table 2).
The authors indicate in Table 2 that some experiments were summarized together. It seems that the specific comparison being made in this section could benefit from some additional repeated trials to see whether the differences in annealing temperatures reproducibly result in a difference in phenotype recovery.
-
We also tested 1300 wild-type PS1159 for spontaneous twitching behavior in water and 1% nicotine for 10 minutes to analyze if this phenotype could frequently occur in a wild-type population, but could not observe any.
While the reported phenotypes (motility and survival) are convincing, if the authors performed such experiments, it might be helpful to know whether any sham injections (buffer only, Cas9 only, guide RNA only) are able to produce any offspring with the uncoordinated phenotype.
-
During the first experiments, we were only able to isolate a few worms with a twitching phenotype and injection results were thus unsatisfactory and not consistent.
Would the authors be able to place the efficiency in the context of existing literature for C. elegans or P. pacificus, for example?
-
After 12 days (generation time is 8-10 days in Panagrolaimus; Schiffer et al., 2019), 16 F1 animals showing reduced crawling activity and a twitching phenotype in water were transferred into single drops of Plectus nematode growth medium (PNGG; supplementary Table S1)
It would be helpful to explicitly state the values shared in Table 4 within the text to provide readers some context about the efficiency of recovering a phenotype.
-
To prepare injection needles using a puller (Sutter Instruments, P-2000), we use a 1-lined so called “bee stinger” program (Oesterle, 2018) as a basis for a 3-lined program (table 1), producing sharper needles with a short taper.
If available, it might be useful for others familiar with nematode injection if an image of this needle compared to a standard needle could be included in the supplement.
-
A cladogram showing the position of the two main species in this study, Panagrolaimus sp. PS1159 and Propanagrolaimus sp. JU765, in relation to the nematode model organisms C. elegans and P. pacificus, as well as A. rhodensis and S. ratti, species with reportedly successful use of the CRISPR system
It might be nice to indicate which species have the features/ methods of interest already developed. For example, you could have a number of columns to the right of the cladogram with a colored circle for:
a) specific biological features, such as life history (parthenogenesis vs. hermaphrodism); b) whether or not RNAi methods have been demonstrated; c) whether or not CRISPR/Cas9 methods have been demonstrated.
This could be a nice way to put the findings in this manuscript into a broader context with a figure.
-
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