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. Author manuscript; available in PMC: 2019 Dec 1.
Published in final edited form as: Chromosoma. 2018 Jul 12;127(4):405–420. doi: 10.1007/s00412-018-0677-6

Fig. 6. Precise integration into target chromosome (PITCh).

Fig. 6.

Two regions of 5-25 base homology at the genomic target site and flanking the gene of interest in the donor plasmid are shown (blue, green) In version 2 of CRIS-PITCh, CRISPR sgRNA cuts adjacent to the microhomology regions in the plasmid, whereas a different sgRNA cuts between the two blocks of microhomology in the genome. The MMEJ pathway (a subset of a-NHEJ) uses the blocks of microhomology to insert the gene of interest into the desired genomic site. Note that the two blocks of microhomology are separated in the end product of PITCh so that the guide RNA that initially cleaved the genomic DNA cannot subsequently cleave the genomic end product. Similarly, the target sites for the PITCh guide RNAs are destroyed in the integrated product. The single dotted or solid lines represent dsDNA of genomic or plasmid DNA, respectively.