Typing CRISPR Systems

By Alyssa Shepard

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A cartoon depiction of cytidine base editing. A base editor, consisting of a cytidine deaminase fused to Cas9, is shown binding to DNA using its guide RNA. The guide RNA base pairs to target DNA, leaving the opposite strand of DNA free to be contacted by the cytidine deaminase, which converts a C to a U within this single-stranded sequence. This deamination yields DNA with a G:U mismatch without creating a double-strand break. Mismatch repair preserves the edit IF the modified strand is used as the template, converting the mismatched G to an A and yielding a single-base-pair edit.
Diagram of the Cascade complex with gRNA bound to target DNA, after recruitment of Cas3. Cas3 is about to nick the non-target strand.
Graphic showing the workflow of using a pooled AAV CRISPR library in vitro. Step A shows the AAV containing the library with either the gRNA only or gRNA plus Cas9 and infecting cells expressing Cas9 or wild-type. The different guides are represented by different colors in individual cells. Step B shows the results of the positive or negative selection, with only one type of guide (color) being chosen. Step C shows verification using next generation sequencing.
A cartoon of a prime editor with two different edit sequences. The DNA sequences are shown with one strand edited and a 5′ DNA flap, before heteroduplex resolution and DNA repair.  The first edit has an unchanged PAM. This DNA is shown connected to the prime editor by a two-way arrow, indicating that the editor can re-bind. Re-nicking is represented by scissors and would remove the newly edited DNA.  The second edit has an altered PAM. A one-way arrow leads from the prime editor to this edit, indicating that the changed PAM prevents the editor from re-binding.
A cartoon overlayed on several crystal structures showing the parts of the prime editor: the Cas9 nickase domain, the reverse transcriptase domain, and the pegRNA.
Alt text: A schematic illustration of the CRISPR/Cas9-based approaches mentioned above. The first illustration (A) shows a DSB made at a transcription start site upstream of an exon. The second illustration (B) shows a DSB made at splice sites. The third illustration (C) shows a DSB made upstream and downstream of an exon to fully remove a genomic fragment. The fourth illustration (D) shows a DSB made within an exon to insert a synthetic polyadenylation signal. 
CRISPR prime editing schematic.

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