CRISPR-Cas9 Targeting of PpAPT in Physcomitrella

The pAct‐Cas9 plasmid used in this study contains a Cas9 expression cassette containing the rice actin 1 promoter and a codon‐optimized version of Cas9 from Streptococcus pyogenes fused to a SV40 nuclear localization (Mali et al., 2013). The pAct‐Cas9 plasmid was constructed as follows: the hCas9 plasmid (plasmid#41815 from AddGene) was digested by NcoI and PmeI and the hCas9 gene was ligated to the pCOR104‐CaMVter plasmid (Proust et al., 2011) previously digested by NcoI and SmaI.
Two sgRNA expression cassettes were designed, each containing a U6 promoter from P. patens, the 5′‐G‐N₍₁₉₎‐3′ sequences targeting PpAPT and the tracrRNA scaffold (Mali et al., 2013; Figures 1 and S1). P. patens genomic sequence for the U6 gene (coordinates 5050300–5050958 on chromosome 1) was identified by Basic Local Alignment Search Tool (http://www.phytozome.net/physcomitrella_er.php) using the Arabidopsis U6‐26 snRNA sequence (X52528; Li et al., 2007) as query. U6 promoter sequence coordinates used for gRNA expression are 5050300–5050621 on chromosome 1. For the design of CRISPR‐Cas targets in the PpAPT gene, both strands of the P. patens adenine phosphoribosyltransferase gene (PpAPT, Phytozome # Pp3c8_16590) were searched using the CRISPOR, free software (http://tefor.net/crispor/crispor.cgi), for sequences of the form 5′‐G‐N(18 or 19)NGG‐3′ with respect to the U6 promoter and Cas9 specificity conditions. Two target loci were selected, one in exon 5 (sgRNA#1) and one in exon 3 (sgRNA#2) of the PpAPT gene (Figure 1). The sgRNA1 and sgRNA2 cassettes were synthesized as gBlocks^® by IDT (www.idtdna.com), PCR‐amplified and introduced into pCR^®II‐TOPO^® TA‐cloning vectors (www.lifetechnologies.com) to give the plasmids psgRNA#1 and psgRNA#2.
Two donor DNA cassettes were used for gene targeting experiments. The PpAPT‐KO4 knockout cassette used for gene targeting experiments bears a 715‐bp 5′ targeting fragment (coordinate 772–1486 on Pp3c8_16590 in Phytozome) and a 702‐bp 3′ targeting fragment (coordinate 1487–2188 on Pp3c8_16590 in Phytozome) of the PpAPT gene, flanking a pAct :: hygroR cassette from the pActHygR plasmid. The pActHygR carries a HPH gene for resistance to hygromycin (Bilang et al., 1991) in fusion with the rice actin 1 promoter from pCOR104 (McElroy et al., 1991) and before a NOS terminator. The 5′ and 3′ sequences of the PpAPT gene present in the PpAPT‐KO4 cassette are flanking the predicted CRISPR‐mediated DSB for target#1 sequence (coordinate 1468–1487 on Pp3c8_16590 in Phytozome). The PpAPT‐KO7 knockout cassette bears a 743‐bp 5′ targeting fragment (coordinate 156–898 on Pp3c8_16590 in Phytozome) and a 778‐bp 3′ targeting fragment (coordinate 917–1694 on Pp3c8_16590 in Phytozome) of the PpAPT gene, flanking a 35S :: neoR cassette from pBNRF for resistance to G418 (Schaefer et al., 2010) cloned in a pCR^®II‐TOPO^® TA‐cloning vector. The 5′ and 3′ sequences of the PpAPT gene present in the PpAPT‐KO7 cassette are flanking the predicted CRISPR‐mediated DSB for the target#2 sequence (coordinate 890–909 on Pp3c8_16590 in Phytozome).