Development of precise CRISPR genome editing and beyond

Prime editing has gained recognition as a highly precise genome editing method, often outperforming other Cas9-based techniques. We recently introduced innovative nCas9-DNA polymerase fusion proteins (DOI: 0.1101/2024.07.09.602710), which create chimeric oligonucleotide-directed editing (CODE) systems designed for targeted "search-and-replace" genome modifications. After extensive engineering with testing over 26 purified variants, we developed two efficient editors, CODEMax and CODEMax(exo+), that achieve accurate genome edits in human cells with minimal off-target effects. Both systems incorporate a modified Bst DNA polymerase with strong strand displacement activity, while CODEMax(exo+) also includes a 5’ to 3’ exonuclease function to facilitate more effective strand invasion and repair, ensuring the desired edits are successfully integrated. Our results show that CODEs perform small insertions, deletions, and substitutions with greater accuracy compared to PEMax across various loci. Ultimately, these CODE systems complement existing prime editors, expanding the toolkit for genome manipulation without inducing double-stranded breaks. 

Beyond CODE, we are engineering a range of CRISPR-based tools to not only improve precise genomic surgeries in vivo but also to advance clinical diagnostics. Some of these technologies like PICNIC (DOI: 10.1101/2024.05.02.24306194) can expand the range of sequences that can be targeted in the genome from ~1% to ~100% by eliminating the need for a PAM.

Learning Objectives:

• Recognize the need for a simple and efficient gene correction technology

• Identify the limitations of existing genome editing technologies in terms of efficiency and off-target effects.

• Describe how CRISPR/Cas systems can be coupled with engineered DNA polymerases to achieve precise modification of any genomic target.