Methods for selecting variant guide rna and prime editing guide rna scaffolds, guide rna and prime editing guide rna compositions and methods of using the same

A method of generating a guide nucleic acid (gRNA) capable of binding a Cas protein, the method comprising:

• a. generating a RNP complex pool by combining a Cas protein with a gRNA having a conserved target region and a randomized scaffold region;
• b. introducing a target DNA bound to or capable of binding to an affinity reagent, wherein the DNA comprises a PAM site and sequence complementary to the conserved target region of the gRNA,
• c. mixing the RNP complex with the affinity reagent and target DNA to generate an RNP-DNA-affinity reagent mixture,
• d. separating an RNP-DNA-affinity reagent complex from the DNA-affinity reagent mixture, and
• e. harvesting the gRNA from the RNP-DNA-affinity reagent complex.

A method of enriching recovery of edited sequences after prime editing comprising:

• (a) generating a ribonucleoprotein (RNP)-DNA complex by contacting at least one prime editing guide RNA (pegRNA) with a target DNA and a prime editing protein complex comprising a Cas9 nickase and a reverse transcriptase (RT), wherein the pegRNA comprises a 5′ region and a 3′ region complementary to the target DNA and an intended edited region to be incorporated into the target DNA the reverse transcript;
• (b) incubating the RNP-DNA complex to allow the Cas nickase to generate a nick in the target DNA and the RT to incorporate the complement of the intended edit into the nicked strand of the target DNA to generate a single-strand DNA incorporated edit;
• (c) introducing a nucleic acid probe sequence complementary to the single-strand DNA incorporated edit to bind to the edited DNA, wherein the probe is labeled or is capable of binding to a label;
• and
• (d) separating the edited DNA by selecting for the label and thus enriching for recovery of edited sequences.