Methods

High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome

Reagents

All chemicals were purchased from Sigma-Aldrich (St. Louis). All enzymes were purchased from New England Biolabs (Beverly, MA). All synthetic DNAs were purchased from Integrated DNA Technologies.

C. elegans strains

C. elegans strains were cultured using standard methods (Brenner, 1974) on nematode growth media (NGM) feeding on OP50 or HB101 bacteria. Animals were maintained at 15°C, 18°C, and 20°C. A list of worm strains created in this study can be found in Table S3.

Molecular biology and cloning

A list of plasmids used in this study can be found in Table S4. All novel plasmids were generated using standard molecular biology techniques. Annotated sequences for all plasmids used in this work are included in Supplemental File 1.

General Injection Procedures

All injections were conducted into young adult (<24 hours) hermaphrodites reared at 15°C on either OP50 (wild type worm strains) or HB101 (unc-119 mutant worm strains). For plasmid injections, plasmids were purified using the Purelink kit (Thermofisher). Unless noted otherwise, the final total concentration of all injected plasmids was 100 ng/µl. Arrays were marked by inclusion of either 1xRed co-injection markers 2 ng/µl pCFJ90 (Pmyo-2::mCherry), 4 ng/µl pGH8 (Prab-3::mCherry) + 4 ng/µl pCFJ104 (Pmyo-3::mCherry) or 1xGreen co-injection markers2 ng/µl pCFJ91(Pmyo-2::gfp) + 8 ng/µl pCFJ421 (Peft-3::gfp::h2b). For CRISPR injections with a single plasmid containing both the repair template and guide RNA expression cassette, the plasmid was included at 65 ng/µl. For CRISPR injections with the repair template and guide RNA expression cassette on different plasmids, the repair template was included at 60 ng/µl and the guide RNA expression plasmid was included at 30 ng/µl. When included, Cas9 expression plasmids were included at 30 ng/µl (cDNA) or 2 ng/µl (with smu-2 introns); higher levels of the germline-licensed Cas9 transgene were toxic. If needed to bring injection mix concentrations to 100 ng/µl, pBluescript(sk+) was used as “stuffer” DNA. After injection, P0s were either singled to fresh OP50 plates (when quantifying event frequency per injected P0) or pooled in groups of 3 – 5 (for construction operations) and incubated at 25°C for 3 – 10 days prior to screening.

Transposon-mediated Cas9 insertion

All MosSci injections were conducted with young adult EG6249 worms. MosSci injection mixes contained 25 – 40 ng/µl ttTi5605-targeting vector, 50 ng/µl pCFJ601 (Peft-3::Mosase), 10 ng/µl 1xRed markers, and 10 ng/µl pMA122 (Phsp-16.41::peel-1) (95 - 110 ng/µl total). MiniMos injections were conducted with young adult EG9814 unc-119(ox819) III worms. MiniMos injection mixes contained 50 ng/µl pCFJ601 (Peft-3::Mosase), 40 ng/µl pMLS714 (miniMos targeting vector), and 10 ng/µl 1xRed markers. Initial MosSci inserts (oxSi---- and oxSi1091) were built using the standard pCF150 targeting vector. To allow removal of the Cbr-unc-119 removal after transgene insertion, we built general targeting vectors for MosSci at the ttTi5605 locus (pMLS640) and for MiniMos (pMLS713) containing a lox2272-_flanked _Cbr-unc-119 cassette. In all cases, genomic inserts were identified by screening for Unc-119+ animals that lacked extrachromosomal array markers. The floxed Cbr-unc-119 was removed from strains by injecting an array containing 50 ng/µl pDD104 (Peft-3::Cre) and selecting for unc F2 progeny.

Cas9 locus modification

Cas9+, unc 119-_animals were injected with 90 ng/µl pMLS719 (miniMos-targeting sgRNA), 10 ng/µl either pMLS716 (P_hsp-16.41::Cre + lox2272 flanked Cbr-unc-119) or 2 ng/µl pMLS791 (Phsp-16.41::Cre + Pmyo-2::2x-nls-cyOFP + lox2272 flanked Cbr-unc-119) + 10 ng/µl 1xRed marker. Cas9 activity was provided by the integrated Cas9 transgene. Injected P0 were incubated at 25°C for 10 days. Inserts were selected by unc-119 rescue and the absence of fluorescence array markers.To remove the Cbr-unc-119 marker, larval animals were heat shocked at 32°C for 2 hours. _Unc-119-_animals were selected by phenotype from the F1 progeny of heat-shocked animals.

Measuring Cas9 activity with dpy-10 sgRNA

For initially screening MosSci Cas9 strains, worms were injected with a low concentration of dpy-10 sgRNA plasmid to improve our dynamic range: 10 ng/µl pMLS597 (PU6::sgRNA(dpy-10)), 10 ng/µl 1xRed markers, and 80 ng/µl stuffer DNA. For screening miniMos-based Cas9 strains, worms were injected with 90 ng/µl pMLS597 (PU6::sgRNA(dpy-10)) and 10 ng/µl 1xRed markers. The higher concentration of pMLS597 resulted in a higher rate of germline mutagenesis of dpy-10 among array(+) animals (not shown). Injected P0 animals were pooled on fresh OP50 plates and incubated at 25°C for 2 – 3 days. Array(+) F1 animals were selected by fluorescence from the co-injection markers. Selected worms were later scored for Dpy and Rol phenotypes under white light.

Measuring tag (GFP) insertion frequencies

For quantifying insertion frequency at multiple target loci in EG9747, worms were injected with 65 ng/µl targeting vector, 10 ng/µl 1xRed markers, and 25ng/µl suffer DNA. For quantifying insertion frequency in miniMos insertion strains, worms were injected with 90 ng/µl targeting vector (pMLS338) and 10 ng/µl 1xRed markers. We used only 65 ng/µl targeting vector when quantifying insertion frequency in EG9747 so that the targeting vector concentration in these injections matched the targeting vector concentration used for quantifying insertion frequency at these sites using plasmid-borne Cas9 (Schwartz and Jorgensen, 2016; data reproduced with permission). After injection, P0 animals were singled to OP50 plates and incubated at either 25°C or room temperature (20°C) until starvation. Starved plates were inspected for array+ and GFP+ animals. Insertion frequency was calculated as (number of plates with GFP + worms) / (number of successful injections), where a ‘successfully injection’ is an injection resulting in array+ worms in the F2 generation.

Quantifying floxed marker excision rates

For quantifying Cre activity, we used unc-32::GFP(Cbr-unc-119+) alleles created in either the EG9886, EG9875, or EG9880 background. Cohorts of ∼20 homozygous young larvae (L1 – L2) of each strain were either incubated at room temperature or at 32°C for 2 hours (“heat shock”). After adulthood, eggs were collected from each cohort, and each hatched animal was scored for the presence of Unc-119(-) phenotype.

Article TitleHigh-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome

Abstract

Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple GFP insertions, 60 – 100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits.

Author Summary Germlines have evolved specialized mechanisms to protect themselves from invasions by transposons and viruses, which create barriers to genome editing techniques. For example, transgenes are silenced in the germline of the nematode C. elegans, thereby creating a barrier to CRISPR editing by Cas9. To facilitate gene editing, we built a collection of C. elegans strains in which Cas9 is never silenced. CRISPR is significantly more efficient in these animals, decreasing the effort researchers need to expend to get edited animals. The strains are available in multiple genetic backgrounds, and contain accessory transgenes to simplify downstream genetics. Together, these strains enable efficient, low-cost genome editing in C. elegans.


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