Construction of gRNA-CRISPR/Cas9 Plasmids

All the sequences for the paired single-stranded DNA oligonucleotides are listed in Table 1. Single-stranded DNA oligonucleotides for gRNAs of SpCas9 were designed following guidelines in GeneArt CRISPR Nuclease Vector Kit (1750624, Invitrogen, Carlsbad, CA, USA) and synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA). Potential off-targets were predicted by recently published software.72 Only gRNAs that had the fewest targets in the coding region were selected for screening of targeted deletion. gRNA for SpCas9 was inserted into a CRISPR nuclease vector containing OFP according to the manufacturer’s protocol. Plasmid DNA was sequenced using U6 forward primers. Single-stranded DNA oligonucleotides of gRNAs for SaCas9 with a BsaI overhang were synthesized by IDT. To increase the cutting efficiency, guanosine nucleotide was added to the 5′ end. The annealed double-stranded oligonucleotides were cloned into the BsaI site of the PX601 plasmid (61591, Addgene). The SpCas9 nickase vector was obtained from Sigma (CAS9D10AP-1EA).

gRNA Sequences and Location within the 3 UTR