Sgrna cloning vector

Plasmids expressing interfering shRNA were generated to suppress endogenous C11orf46 protein expression utilizing the pSUPERIOR.puro vector system (Oligoengine). Their target sequences are: C11orf46 shRNA-1 with strong suppression; 5′-CAAACTGAATTTGCTCCAGAA- 3′ and C11orf46 shRNA-2 with milder suppression; 5′- GAAGACAGCTTGTACCTGGTT- 3′. A scrambled sequence that shows no homology to any known messenger RNA was utilized to produce the Control shRNA (5′-ATCTCGCTTGGGCGAGAGTAAG-3′). The HA and scFv-tagged RNAi resistant wild-type human C11orf46 expression constructs containing four silent mutations (underlined) in the target sequence of C11orf46 shRNA (5′- CAAACAGAGTTCGCACCAGAA-3′) were produced and cloned into CAG promoter driven plasmid (pCAGGS1vector) for rescue experiments. dCas9-SunTag coding sequence was also transferred into pCAGGS1 vector. Two single guide RNA sequences each Dclk1 or Sema6a promoter region were using online CRISPR design tool^{67 (link)}. Those protospacer sequences were cloned into sgRNA cloning vector (Addgene #41824) with the protocol distributed on the addgene website (https//media.addgene.org/data/93/40/adf4a4fe-5e77-11e2-9c30-003048dd6500.pdf). The protospacer sequences are listed in Supplementary Table 5. CAG promoter driven mouse Sema6a expression plasmid (CAG-mSema6a) was cloned using Sema6a (NM_018744) Mouse Tagged ORF clone (ORIGENE, Cat#MR211486).

The sgRNA cloning vector was obtained from Addgene (Plasmid #41824). sgRNA sequences were selected using CRISPRdirect (http://crispr.dbcls.jp)^{20 (link)}. The selected sequences were sgRNA1 (5′-GAGACCATCTACTCTTGCCTTGG-3′), sgRNA2 (5′-AGATAATTACCATGCAACTTGG-3′), and sgRNA3 (5′-GGGTCACACAGAAGACGCCAGG-3′) where proto-spacer adjacent motif (PAM) sequences are underlined. sgRNAs were prepared as previously described^{6 (link)}. sgRNA sequences except the first nucleotide and PAM were cloned into sgRNA cloning vectors by inverse PCR using primer pairs sgRNA1F/sgRNA1R for sgRNA1, sgRNA2F/sgRNA2R for sgRNA2, and sgRNA3F/sgRNA3R for sgRNA3 (Supplementary Table 1). sgRNA fragments were amplified using T7-added primers (T7sgRNA1F for sgRNA1, T7sgRNA2F for sgRNA2, and T7sgRNA3F for sgRNA3) and sgRNA-R (Supplementary Table 1). sgRNAs were synthesized by means of an in vitro transcription reaction using the mMESSAGE mMACHINE T7 Transcription Kit (Ambion). Transcribed RNAs were purified using the MEGAclear RNA Kit (Ambion).