CRISPR Guide Update

By Alyssa Shepard

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Schematic of Fragmid assembly process from day one to day four and resulting vector architecture.  On day one fragment plasmids plus a destination vector are assembled using Golden Gate (BbsI). On day two the assembled vector containing a Guide (2xBsmBI), Promoter, N-terminus, Cas protein, C’terminus, and 2A-Selection undergoes an exonuclease V cleanup followed by transformation and plating. On day three two colonies per construct are picked, miniprepped and restriction digested for gel validation/whole plasmid sequencing. Below the assembled vector are examples of six vector architectures including pRDA_512 lentivirus, pRDA_722 lentivirus, pRDA_789 CROPseq lentivirus, pRDA_889 AAV, pRDA_575 Piggyback, and pRDA_791 empty plasmid.
Overview of the parts of CRISPR. The bacterial chromosome encodes a tracrRNA (in some systems including Cas9), Cas proteins, and a CRISPR array. The CRISPR array is composed of identical repeat sequences and variable spacer sequences. The array is transcribed and processed into crRNAs, each including one repeat and one spacer. In bacteria, these crRNAs are bound by Cas proteins (Cas9 shown here). The repeat sequence base pairs with the tracrRNA, and the spacer sequence is used to target complementary DNA sequences. In laboratory settings, an sgRNA includes the crRNA and tracrRNA sequences in a “single-guide RNA” that performs both functions. Cas9 cuts both the target and nontarget DNA strands upstream of the PAM site found in the nontarget strand.
screenshot of various PRIDICT webpages
Cartoon summary of Cas9 activity.
Graphic representation of the FITS screening process

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