• CRISPR-Cas12a
• microhomology-mediated end joining
• precise DNA replacement

The development of more efficient and precise methods to engineer the cellular genome is an essential aim in synthetic biology, life sciences and medicine. The available approaches have many important limitations, like the requirement of bulky homology arms surrounding the donor DNA, the frequent presence of unwanted indels at the donor DNA-target locus junction and complex genome editing design with a high risk of collateral DNA damages.
I have established a simple and efficient method based on CRISPR-Cas12a for precise microhomology-mediated targeted DNA replacement in human cells, with the following advantages: it uses the naturally high-fidelity CRISPR-Cas12a nuclease and down to only 2 different guide-RNAs to limit off-target genome editing events, requires the simple engineering of donor DNA with short microhomologies (15-30bp) with the target locus to produce scarless junctions at genome integration sites and exploits the transient use of inhibitors of non-homologous end joining kinase DNA-PK to prevent off-target donor DNA integration and further minimise genome surgery errors. The precision of CRISPR-Cas12a for microhomology-mediated DNA replacement is as high as using long (1kb) homology arms, while it outperformed strategies based on homology-independent targeted DNA integration and reduced the presence of indels at the donor DNA-genome junction by 2-6 folds if compared to microhomology-mediated approaches using CRISPR-Cas9. The developed CRISPR-Cas12a microhomology-mediated DNA replacement strategy paves the way for all the biotechnological and medical applications where precise and efficient genome manipulation is essential.