Journal: Molecular Biomedicine

Article Title: Random-PE: an efficient integration of random sequences into mammalian genome by prime editing

doi: 10.1186/s43556-021-00057-w

Figure Lengend Snippet: Workflow of Random-PE in cultured mammalian cells. a . Diagram showing the design and screen of functional PE3 system targeting interest genes. b . Construction of PCR pegRNA random library. A forward primer located upstream of U6 promoter and a reverse primer containing complementary sequences to the sgRNA scaffold, HA, aimed random sequences, PBS, and U6 termination signal are used to amplify the existing sgRNA or pegRNA. c . Delivery of pegRNA library together with PE2 and nick sgRNA plasmids into interest cells. d . Detection of the desired insertion of random sequences in the genome. Genomic DNAs are extracted from transfected cells and subjected to PCR amplification using primers flanking the target region. The presence of the desired insertion of random sequences is detected by Sanger sequencing and quantified by HTS

Article Snippet: The plasmid PE2 was obtained from addgene (#132775).

Techniques: Cell Culture, Functional Assay, Transfection, Amplification, Sequencing

Journal: Cell

Article Title: Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins

doi: 10.1016/j.cell.2021.12.021

Figure Lengend Snippet:

Article Snippet: Lentiviral vectors were constructed via USER cloning into the lentiCRISPRv2 backbone (Addgene #135955).

Techniques: Virus, Recombinant, Transfection, SYBR Green Assay, DNA Extraction, Multiplex Assay, Purification, Gel Extraction, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Cell Isolation, Titration, Amplification, Sequencing, Software

Journal: bioRxiv

Article Title: PROTAC molecule-mediated SpCas9 protein degradation for precise genome editing

doi: 10.1101/2025.01.07.631496

Figure Lengend Snippet: A .Optimized CASPROTAC 6 degraded dCas9 proteins. 293T-Tet-On 3G cells with stable integration of dCas9-APEX2 were pretreated with doxycycline (500 ng/mL) for 9 h to the simultaneous expression of Cas9-APEX2 protein complex, 15 hrs after removing doxycycline, CASPROTAC 6 was added for 24 hrs in a dose manner. The protein level of dCas9-APEX2 was monitored by Western blotting. B . Quantification of the intensity of the staining from A. Anti-Flag antibody was used to detect the level of dCas9-APEX-Flag proteins. Anti-β-actin was used as the loading control. C . The 293T-nCas9-PE2 bulk cells were pretreated with doxycycline (500 ng/mL) for 24 h, then removing doxycycline, CASPROTAC 6 was added for 24 hrs. The protein level of nCas9-PE2 was monitored by Western blotting. D . Quantification of the intensity of the staining from C. Anti-Cas9 antibody was used to detect the level of dCas9-APEX-Flag proteins. Anti-β-actin was used as the loading control. E . 293T-nCas9-BE3RA bulk cells were pretreated with doxycycline (500 ng/mL) for 24 h. Then, after removing doxycycline, CASPROTAC 6 was added for another 24 hrs. The protein level of nCas9-BE3RA was monitored by Western blotting. F . Quantification of the intensity of the staining from E. Anti-Cas9 antibody was used to detect the level of dCas9-APEX-Flag proteins. Anti-β-actin was used as the loading control. Bars show mean value□±□s.e.m. and significance was calculated using Student’s t-test (n = 2 or 3). *p < 0.05, **p < 0.005, and ***p < 0.0001 (versus the control). G . CASPROTAC 6 reduced CRISPR editing efficiency. FANCF-gRNA and SpCas9 protein were mixed at a 1:1 molar ratio (20 pmol SpCas9+ 20 pmol FANCF-gRNA for 1 million cells) and incubated at room temperature for 20 min to allow the formation of the CRISPR ribonucleoprotein complexes. 293T cells were electroporated with FANCF-RNP. Six hours after electroporation, cells were incubated with CASPROTAC 6 for 24 hrs. CRISPR editing efficiency was measured by amplicon sequencing. H . The reduction of CRISPR/SpCas9-mediated editing based on the amplicon sequencing results. I . Batch output mode comparing CRISPR editing of control and CASPROTAC 6-treated cells. Each column represents the base composition of a single nucleotide at the FANCF site 2 locus. The percentage of each base or indel at each nucleotide is shown as a proportion of each row (n=1). *p < 0.05, **p < 0.005, and ***p < 0.0001 (versus the control).

Article Snippet: For 293T-nCas9-PE2 bulk cells, the wild-type 293T cell line was grown in 6 well plates (50% confluency) and co-transfected with 1.0 μg Xlone-PE2 (Addgene 136463) plasmid and 1.0 μg PiggyBac Transposase vector 6 µg PEI (Polyscience, 23966) in 50 µl of serum-free medium and selected with 3□μg ml -1 Blasticidin (Biomol) for 2-3 days.

Techniques: Expressing, Western Blot, Staining, Control, CRISPR, Incubation, Electroporation, Amplification, Sequencing