To build dCas9-GFP1114x, we first synthesized two fragments of gBlocks, each containing seven copies of the coding sequence of GFP11 tag. Then we modified the SunTag vector (pHR-dSV40-NLS-dCas9-24xGCN4_v4-NLS-P2A-BFP-dWPRE, addgene #60910) to replace 24xGCN4 with GFP1114x. The ...
pHRdSV40-NLS-dCas9-24xGCN4_v4-NLS-P2A-BFP-dWPRE pU6-(BbsI)_CBh-Cas9-T2A-BFP-P2A-Ad4E1B p426-SNR52p-gRNA.CAN1.Y-SUP4t pCLIP-Cas9(D10a)-Nickase-hCMV-Puro pHRdSV40-scFv-GCN4-sfGFP-VP64-GB1-NLS pU6-(BbsI)_CBh-Cas9-T2A-mcherry-P2A-Ad4E4orf6 pAAV-Guide-it-1 Vector ...
f.为了多至30x106的电穿孔,使用1ml lv kit l unit(目录号#:v4lc-2002)。 [0092] g.为了多至100x106或更多的电穿孔,使用1ml lv kit l unit(目录号#:aaf-1002l)。 [0093] h.通过将总细胞数除以5x106然后乘以步骤3中的最终rnp复合物数量来计算所需的rnp复合物总量。 [0094] i.示例:如果细胞数为...
Here, we present the method called “primary T cell crisprQTL,” a high-throughput, single-cell, pooled CRISPRi-based functional screening framework to map non-coding regulatory elements to genes in primary human CD4+T cells. We perturbed transcription start sites (TSSs) and putative regulatory ...
For piggyBac transposition of fusion protein constructs, cells were electroporated with ~210 ng of AsCas12a fusion protein plasmid and ~84 ng of Super PiggyBac Transposase Expression Vector (PB210PA-1, Systems Biosciences) using the SF Cell Line 4D-Nucleofector X Kit (V4XC-2032, Lonza ...
pyogenes Cas9/gRNA complexes were prepared in vitro, mixed with HDR donor templates, and electroporated into WTCGFP 1–10 cells by nucleofection using P3 solution (Lonza #V4SP-3096) and the CA-137 program. Nucleofected cells were plated in StemFlex media (Thermo Fisher Scientific #A3349401)...
For the generation of the Scyl1AIv4 allele, the following HDR template encoding the AIv4 cassette sequence to be inserted and homology arms of 58 and 60 nucleotides (lower case) was used: agatcgtgaaagccctcagcttcctggtcaacgactgcaacctcatccacaataatgtGTAAGTAATAACTTCGTATAGCATACATTATACGAAGTTATTCAAGGT...
The 5′ tag region (G(−15)–C(−1)) of the crRNA adopts a single-stranded conformation and is extensively recognized by Cas7.1 and Cas7.2 (Figures 3A, 3B, 4A, and S5). The thumb-like β-hairpin of Cas7.1 intercalates between A(−2) and G(−4), resulting in the base ...
Reverse transcription of gRNAs and adapter addition was achieved using a custom protocol with the Clontech SMART-Seq v4 Ultra Low Input RNA sequencing kit (634894). Approximately 30 ng of RNA from YFP+CMs or 500 ng RNA from input CMs was reverse transcribed as directed in the manufacturer...
The CRISPR-Cas mining pipeline CRISPRCasTyper (https://github.com/Russel88/CRISPRCasTyper/releases/tag/v1.8.0)21 was utilized to detect known CRISPR-Cas systems since this pipeline annotates Cas proteins using the most recently updated Cas HMM profile database, and it has established a reasonab...