(a) Structure of wild-type I-CreI. The two I-CreI monomers comprising the functional homodimer are shown in gray. The primary amino acids involved in conferring target site specificity are colored red in the structure. The recognition sequence for I-CreI is shown beneath the structure with base pairs that are directly contacted by the enzyme shown in red. (b, c) Models of the single-chain (b) M1PCSK9 and (c) M2PCSK9 meganucleases and their intended target site in the PCSK9 gene are shown. The N- and C-terminal subunits of each meganuclease are shown in gray. Amino acids comprising the DNA-binding surface are colored teal and identified with numbering consistent with wild-type I-CreI. The PCSK9 target sequence is shown with base pairs that are believed to be contacted directly by the engineered meganucleases colored in teal. The three amino acids that differ between M1PCSK9 and M2PCSK9 are colored blue, as are the two base pairs in the target sequence that these amino acids are predicted to contact. (d) M1PCSK9 and M2PCSK9 introduce mutations at the intended site in PCSK9 in HEK-293 cells. Cells were mock transfected or electroporated with mRNA encoding either M1PCSK9 or M2PCSK9. Seventy-two hours post transfection, genomic DNA was isolated and amplified using PCSK9-specific PCR primers. PCR products were digested with T7-endonuclease and visualized on an agarose gel. The PCR products from meganuclease-transfected cells yielded smaller bands (indicated by black arrows) consistent with a high frequency of indel mutations at the intended target site. Deep sequencing of the PCR products revealed indel frequencies of 54.0% and 56.5% in cells transfected with M1PCSK9 and M2PCSK9, respectively. Similar results were obtained in three experiments. (e, f) In vivo evaluation of editing efficiency of M1PCSK9 and M2PCSK9 on episomal hPCSK9 cDNA delivered by AAV9.hPCSK9 vector in Rag1 KO mice. Adult male Rag1 KO mice first received an intravenous injection of AAV9.hPCSK9 vector (3.5x1010 GC), and 14 days later received a second vector injection of (e) AAV8-M1PCSK9 or (f) AAV8-M2PCSK9 at three doses (5.0x1011, 1.0x1011, or 2.0x1010 GC). PCSK9 levels were measured in mouse serum samples collected on days 7, 14, 21, 28, and 56 and presented as percentage of levels on day 14. Data from each individual mouse are shown (n=3 mice per cohort).