Journal: Molecular Therapy. Methods & Clinical Development
Article Title: Genome-wide Specificity of Highly Efficient TALENs and CRISPR/Cas9 for T Cell Receptor Modification
Figure Lengend Snippet: DSB Repair and Targeted Genome Editing of the TCR Loci Using Designer Nucleases (A) During NHEJ-repair of TALEN- and CRISPR/Cas9-induced DSBs, frameshift mutations can result in gene knockout, or episomal IDLV can be integrated into DSBs, allowing for the permanent marking of off-target DSBs. If donor DNA is provided, HDR can lead to targeted integration of an expression cassette, i.e., therapeutic TCR chains. (B) The TCR α and β locus are composed of a number of variable (V), joining (J), constant (C), and, in the case of TRBC , diversity (D) gene segments (numbers of functional genes from IMGT/GENE-DB 53 version 3.1.16, December 14, 2016). The positions of TALEN and gRNA target sequences for TCR knockout in the TCR α constant region ( TRAC ) and a homologous sequence shared by both TCR β constant regions ( TRBC1/2 ) are marked by scissor symbols. DSB, DNA double-strand break; gRNA, guide RNA; HDR, homology-directed repair; IDLV, integrase-defective lentiviral vector; LTR, long terminal repeat; NHEJ, non-homologous end joining; PAM, protospacer adjacent motif; RVD, repeat variable di-residue.
Article Snippet: Donor Construction and Targeted Integration PCR For the 200 bp α chain donor construct (TA200G), we ordered a GeneArt Strings DNA Fragment (Thermo Fisher Scientific) containing two 200 bp regions flanking the αT4 cutting site in the TRAC locus and restriction sites AsiSI and SbfI for cloning into a lentiviral transfer vector in antisense direction.
Techniques: Non-Homologous End Joining, CRISPR, Gene Knockout, Expressing, Functional Assay, Knock-Out, Sequencing, Plasmid Preparation