Journal: Scientific Reports
Article Title: Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric fusion proteins
doi: 10.1038/s41598-021-95406-8
Figure Lengend Snippet: Marked increase in indel efficiency of the Cas9-RecJ and Cas9-GFP chimeric fusion proteins. ( A ) Schematic of a translational fusion of a modifier protein to SpyCas9. His tag (6X His) for protein purification and nuclear localization signal (NLS) were placed. ( B ) Comparison of the DNA cleavage activity of SpyCas9 and its chimeric proteins, C-terminal chimeric SpyCas9 translational fusion proteins with the nucleases RecJ, RecE, T5, lambda, mung bean exonuclease, and human terminal deoxynucleotidyl transferase (TdT), and with green fluorescent protein (GFP). The modifier proteins are described in Supplementary Table . The CCR5 DNA was digested with the purified enzymes and resolved on an agarose gel. Successful digestion cuts the 1.5 kb substrate DNA into two 0.75 kb DNAs. ( C ) DNA band intensities in the gel image. Statistical comparison was made between C9 and each of the C9R or C9G proteins. Significance was indicated on top of each bar. One-way ANOVA using Tukey’s multiple-comparison test. The fold change relative to C9 is indicated above each bar. ( D ) DNA insertion and deletion (Indel) efficiency of C9, C9R, and C9G for the CCR5 gene in HEK293T cells at 8, 16, and 24 h after RNP transfection as measured by a T7E1 assay. Averages from the three replicate experiments are plotted. Two-way ANOVA using Tukey’s multiple-comparison test. ( E ) Indel efficiency of C9, C9R, and C9G for the CCR5 , HPRT1 , and EMX1 genes (left to right) in HEK293T cells. At 24 h ( CCR5 and HPRT1 ) and 8 h ( EMX1 ) post-transfection, cells were harvested and subjected to targeted deep sequencing. The experiments were performed in triplicate. NT group represent the NGS data from non-treated control. One-way ANOVA using Tukey’s multiple-comparison test was performed using C9 data as control. ( F ) Distribution of the DNA deletion sizes after treatment with C9, C9R, and C9G. The number of deletion events around the protospacer site in the CCR5 gene was counted according to the 15 bp deletion intervals from targeted deep sequencing data (n = 3) data. ( G ) Distribution of DNA insertion sizes after treatment with C9, C9R, and C9G in the CCR5 gene. The number of insertion events was counted according to the 1 bp insertion intervals from targeted deep sequencing data (n = 3) data. 6–10 and 11–61 bp insertion intervals were also shown to include events with smaller numbers. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, and n.s. indicates not significant.
Article Snippet: The SpyCas9 coding sequence (Addgene, #138566) was ligated into a pET28a vector (Merck Biosciences, Darmstadt, Germany) that had been linearized by Nco I and Xho I double digestion.
Techniques: Protein Purification, Comparison, Activity Assay, Purification, Agarose Gel Electrophoresis, Transfection, Sequencing, Control
Addgene inc is a verified supplier 
