Pc0046 ef1a pspcas13b nes hiv

The pC0046-EF1a-PspCas13b-NES-HIV (Addgene #103862) was modified to enable Cas13b endonuclease activity within the nucleus for specific eRNA transcript targeting. This was accomplished by removing the HIV nuclear export signal (NES) and replacing it with the SV40 nuclear localization sequence (NLS). A nucleoplasmin NLS was also inserted immediately upstream of the Cas13b coding sequence. Finally, a T2A self-cleaving peptide and Blasticidin resistance cassette was inserted for the selection of positive clones and the 3× HA tags were conserved. This resulted in the Lenti-PspCas13b-NLS-HA-Blast vector for eRNA degradation. Stable Cas13b expression cells were created for MDA-MB-436 and OVCAR3 cells following the same methods as described in the engineering dCas9-KRAB cells section. The primary antibody for Western blot was anti-HA tag (Sigma, SAB4300603).

For a list of plasmids, we generated for this study, see Additional file 3: Table S2. The original plasmids we used for this project were obtained from Addgene: pCFD3 (#49410), pCFD5 (#73914), pACG:eCFP (#32597), pDsRed-attP (#51019), Ac5-Stable2-Neo (#32426), pC0056-LwaCas13a-msfGFP-NES (#105815), pC0040-LwaCas13a crRNA backbone (#103851), pC0046-EF1a-PspCas13b-NES-HIV (#103862), pC0043-PspCas13b crRNA backbone (#103854), pC0054-CMV-dPspCas13b-longlinker-ADAR2DD (E488Q/T375G) (103870), pXR001: EF1-CasRX-2A-eGFP (#109049), pXR004: CasRX pre-gRNA cloning backbone (#109054), pBID-UASc (#35200), [10 (link), 12 (link), 23 (link), 35 (link), 46 (link), 50 (link), 107 (link)–110 (link)]. We also obtained plasmids from the Drosophila Genetic Resource Center (DGRC): pAFW (#1111), pAHW (#1095), act-PhiC31-integrase (#1368). We also used plasmids we previously generated, enDmC, to generate some constructs for this study [8 (link)]. pMT-Gal4-puro plasmid was a kind gift from Christoph Metzendorf (University of Uppsala). All fragments used for the cloning step were amplified via PCR using Q5 high-fidelity DNA polymerase (NEB #M0491S) (Additional file 4: Table S3) and fused together via Gibson assembly reaction [111 (link)].

Engineered RfxCas13d and PspCas13b coding sequences were cloned into pL7 vectors for cell-specific expression in Purkinje cells [14 (link)] and prepared using the EndoFree Plasmid Maxi Kit (QIAGEN) according to the manufacturer’s protocol. The engineered RfxCas13d sequence was PCR amplified from the pXR001: EF1a-CasRx-2A-EGFP vector (Addgene plasmid #109049). The engineered PspCas13b sequence was PCR amplified from the PspCas13b vector pC0046-EF1a-PspCas13b-NES-HIV (Addgene plasmid #103862), a gift from Feng Zhang.