Plasmids have been deposited in Addgene with the following accession numbers: Cas9-sgRNA plasmid targeting a site near ttTi5605, #47550; Cas9-sgRNA plasmid with no targeting sequence, #47549; Peft-3::Cre::tbb-2 3’UTR construct, #47551. All other plasmids used in this study are available from the authors upon request.
To construct the Cas9-sgRNA expression plasmid shown inFig. 1c , we first designed a synthetic gene encoding Cas9, with C. elegans coding bias and synthetic C. elegans introns, using the C. elegans Codon Adapter40 (link). Our Cas9 sequence includes a Nuclear Localization Signal and an HA tag at the C-terminus. The synthetic gene was produced as a series of overlapping 500 bp gBlocks (Integrated DNA Technologies), assembled using Gibson Assembly (New England BioLabs) and inserted into the vector pCFJ601 (Peft-3::Mos1 Transposase::tbb-2 3’UTR)17 (link) in place of the Mos1 transposase. Next, a gBlock containing the U6 promoter and sgRNA sequence was inserted 3’ of the tbb-2 3’UTR. Genomic targets of Cas9 conform to the target sequence GN19NGG, where N is any base. The initial G is a requirement for transcription initiation by the U6 promoter, and the NGG (PAM) motif is required for Cas9 activity (note that the NGG motif must be present in the genomic target but is not included in the sgRNA sequence). To target Cas9 to different genomic sequences, we inserted the desired targeting sequence into the Cas9 + sgRNA construct using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) with forward primer 5’-N19GTTTTAGAGCTAGAAATAGCAAGT-3’, where N19 is replaced by the desired 19 bp targeting sequence, and reverse primer 5’-CAAGACATCTCGCAATAGG-3’. Supplementary Table 5 lists the targeting sequences used in this study.
Targeting vectors for single-copy transgene insertion on chromosome II were constructed in the pCFJ150 vector backbone20 (link) using Gateway cloning. We used site-directed mutagenesis with the Q5 site-directed mutagenesis kit (New England Biolabs) to delete a short region of the 3’ recombination arm comprising the Cas9 target sequence, to prevent the homologous repair templates from being cleaved by Cas9.
Homologous repair templates for GFP insertion and lin-31 mutagenesis were constructed in two steps. First, we PCR amplified a 3–4 kb region centered on the desired modification from N2 genomic DNA and cloned the resulting fragment into the pCR-Blunt vector using the ZeroBlunt TOPO Cloning Kit (Life Technologies). Second, we modified this genomic clone by inserting GFP (for GFP knock-ins) or a 3’ exon containing point mutations (for lin-31 mutagenesis), along with the unc-119(+) rescue gene flanked by LoxP sites. GFP and unc-119(+) fragments were generated by PCR, and LoxP sites were included in the unc-119(+) primers. The mutated lin-31 3’ exons were synthesized as gBlocks. These fragments were integrated into the genomic clones using Gibson assembly, which allows for seamless fusion of DNA fragments without the need to include any extra sequence (e.g. restriction sites). To avoid cleavage of the repair templates by Cas9, we deleted or mutated the Cas9 target site in all repair templates. Complete plasmid sequences of all targeting vectors are available from the authors upon request.
To construct the Peft-3::Cre::tbb-2 3’UTR plasmid used for removal of selectable markers with Cre recombinase, we first amplified the Cre ORF from the plasmid pEM3 (ref. 41 (link)) and cloned it into the Gateway donor vector pDONR221. We then performed a 3-fragment gateway reaction using our Cre donor vector, pCFJ386 (Peft-3; a gift from Christian Frøkjær-Jensen), pCM1.36 (tbb-2 3’UTR)42 (link) and the destination vector pCFJ212 (ref. 17 (link)), which contains an unc-119(+) rescue gene.
Supplementary Table 6 lists all primers used in this study.
To construct the Cas9-sgRNA expression plasmid shown in
Targeting vectors for single-copy transgene insertion on chromosome II were constructed in the pCFJ150 vector backbone20 (link) using Gateway cloning. We used site-directed mutagenesis with the Q5 site-directed mutagenesis kit (New England Biolabs) to delete a short region of the 3’ recombination arm comprising the Cas9 target sequence, to prevent the homologous repair templates from being cleaved by Cas9.
Homologous repair templates for GFP insertion and lin-31 mutagenesis were constructed in two steps. First, we PCR amplified a 3–4 kb region centered on the desired modification from N2 genomic DNA and cloned the resulting fragment into the pCR-Blunt vector using the ZeroBlunt TOPO Cloning Kit (Life Technologies). Second, we modified this genomic clone by inserting GFP (for GFP knock-ins) or a 3’ exon containing point mutations (for lin-31 mutagenesis), along with the unc-119(+) rescue gene flanked by LoxP sites. GFP and unc-119(+) fragments were generated by PCR, and LoxP sites were included in the unc-119(+) primers. The mutated lin-31 3’ exons were synthesized as gBlocks. These fragments were integrated into the genomic clones using Gibson assembly, which allows for seamless fusion of DNA fragments without the need to include any extra sequence (e.g. restriction sites). To avoid cleavage of the repair templates by Cas9, we deleted or mutated the Cas9 target site in all repair templates. Complete plasmid sequences of all targeting vectors are available from the authors upon request.
To construct the Peft-3::Cre::tbb-2 3’UTR plasmid used for removal of selectable markers with Cre recombinase, we first amplified the Cre ORF from the plasmid pEM3 (ref. 41 (link)) and cloned it into the Gateway donor vector pDONR221. We then performed a 3-fragment gateway reaction using our Cre donor vector, pCFJ386 (Peft-3; a gift from Christian Frøkjær-Jensen), pCM1.36 (tbb-2 3’UTR)42 (link) and the destination vector pCFJ212 (ref. 17 (link)), which contains an unc-119(+) rescue gene.
