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.
AAV infection of retinal neurons.

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