Takara Guide-it Long ssDNA Production System v2 \50 Rxns

The Guide-it Long ssDNA Production System v2 includes all the reagents and materials necessary for on-demand preparation of single-stranded DNA (ssDNA) repair templates of any sequence up to 5,000 nt in length for engineering gene knockins using CRISPR/Cas9 technology or other genome editing tools. It has been demonstrated that ssDNA offers two key advantages over dsDNA templates for precise genome editing applications: greatly reduced toxicity and a much lower likelihood of random or off-target integration. The kit employs a simple and fast method that involves conversion of a dsDNA PCR product into ssDNA via selective digestion of either the sense or the antisense strand. Following digestion, ssDNA products are purified using silica membrane spin columns included with the kit.

Cat. # 632666 includes sufficient quantities of reagents and materials for preparation of 50 ssDNA templates.

Improved processing of challenging templates and production of ssDNA up to 5,000 nt with the Guide-it Long ssDNA Production System v2.The Guide-it Long ssDNA Production System v2 enables successful production of ssDNA ranging from 500 nt to 5,000 nt in length. The gel images show dsDNA substrates of varying length (Lane 1) and corresponding sense (SS) and antisense (AS) ssDNA products (Lane 2) following enzymatic digestion and cleanup with the kit. Each ssDNA HDR template was designed to target theCCR5gene.

Efficient editing and with negligible off-target insertion using ssDNA donor templates. Panel A.Tagging of GAPDH with a fluorescent protein (AcGFP1). An HDR template was designed to fuse theAcGFP1coding sequence in-frame at the C-terminus of theGAPDHgene.Panel B.HEK293 cells were transfected with plasmid, dsDNA, or ssDNA forms of theAcGFP1HDR template, with or without co-transfection of plasmids encoding for Cas9 and an sgRNA targeting theGAPDHlocus. Cells were grown for three days and then analyzed by flow cytometry. While the plasmid template resulted in very little integration as demonstrated by the low percentages of fluorescent cells, the dsDNA yielded significant proportions of AcGFP1-postive cells, even in the absence of Cas9-sgRNA expression, suggesting a significant level of non-specific integration. Both sense (S) and antisense (A) ssDNA repair templates were associated with efficient insertion of theAcGFP1sequence in the presence but not in the absence of Cas9 and sgRNA, suggesting that insertion of theAcGFP1sequence was highly specific.

Knockin of murine T-cell receptor α- and β-chains at theTRAClocus in human CD3+ T cells. Panel A.CRISPR/Cas9 RNPs with sgRNAs targeting bothTRACandTRBCloci were coelectroporated with an ssDNA HDR template encoding murine T-cell receptor α- and β-chains. The HDR template was designed to include 350-nt homology arms targeting exon 1 of theTRAClocus in addition to the following elements: P2A and T2A, self-cleaving peptide inserts;TRBCmouse, mouse TCR-β constant region;TRACmouse, mouse TCR-α constant region; pGHpA, poly-A tail. The total length of the ssDNA was 2,800 nt.Panel B.Flow cytometric analysis of resulting T-cell populations 10 days after editing using antibodies against mouse TCR-β and human TCR-α/β. Cells in the negative control population (nonedited cells) were positive for the expression of human TCRs. For cells electroporated with RNPs targeting bothTRACandTRBCloci, knockout of the endogenous TCR could be detected. In cells coelectroporated with RNPs combined with the ssDNA HDR template, expression of the murine T-cell receptor α- and β-chains could be detected.