scei neb r0694 cas9 mrna matsuwaka  (New England Biolabs)

Journal: Frontiers in Genome Editing

Article Title: The precision strategy of human genome correction via a set of circular donor DNA and its cleaver

doi: 10.3389/fgeed.2026.1718252

Figure Lengend Snippet: Several hundred base pairs in distance between the cleavage site and the designed sequence on a circular donor plasmid are required to retain the designed allele. (A) A circular donor plasmid, pMB1KmHygTK-HPRT(ISCEI(318)Ex2Syn), pMB1KmHygTK-HPRT(ISCEI(545)Ex2Syn), or pMB1KmHygTK-HPRT(Ex2Syn(1195)ISCEI), consists of the vector DNA (pink line) and the homologous DNA (blue line) with the Syn sequence and the ISCEI cleavage site. In the left column, three types of circular donor plasmids depict the length of left homology, the distance between the cleavage point and the Syn sequence, and the length of right homology with a cleavage type (no cleavage, intra-cellular cleavage, or linearized at the ISCEI site). In the following three columns, the numbers show the total cells transfected by electroporation, the whole integration clones observed as hygromycin-resistant (Hyg R ) colonies, the TD clones, and the abortion clones in brackets, which were doubly positive and singly positive in the duplication test , respectively. In the last three columns, the frequency of the TD clones relative to the whole Hyg R colonies, the number of the Syn -retaining TD clones, and the frequency of the Syn -retaining TD clones relative to the TD clones are shown. In the case of the intra-cellular cleavage type, the nls-I-SceI expression plasmid, pCI- nls-I-SceI or pCAG.I-SceI plasmid (bottom on lane v), was co-transfected with a circular donor DNA ( Materials and Methods ). The calculations by CHISQ.TEST ( Excel ) are shown in . (B) The duplication test for the clones derived from the donor plasmid with HPRT(ISCEI(545)Ex2Syn): the results from the duplication test for 12 hygromycin-resistant and 6-thioguanine-resistant (Hyg R TG R ) clones (HTG-A1–A12) out of 1,768 Hyg R clones (top of lane iii in panel (A) ) are shown. Out of 12, 10 clones are doubly positive, 1 clone (HTG-A9) is singly positive (Ab: Abortion), and 1 clone (HTG-A1) is doubly negative in the duplication test. The results for 9 Hyg R TG R clones (HTG-B1–B9) out of 1708 Hyg R clones [Center of lane iii in (A) ] are shown. Out of 9, 6 clones are doubly positive and 3 clones (HTG-B5, B8, and B9) are doubly negative in the test. The results for 10 Hyg R TG R clones (HTG-C1–C10) out of 2,872 Hyg R clones (bottom of lane iii in panel A) are shown. Out of 10, 7 clones are doubly positive and 3 clones (HTG-C1, C3, and C4) are doubly negative in the test. Random integration (RI) test for the clones derived from the donor plasmid with HPRT(ISCEI(545)Ex2Syn): The results from the RI test for HTG-A1–A12, HTG-B1–B9, and HTG-C1–C10 clones are shown, based on the PCR assay with target-5′-outside forward primer 2 (closed blue arrow) and target-3′-outside reverse primer 2 (open blue arrow), which is the same primer pair as that in the pop-out test . TD shows to be doubly positive in the duplication test and negative in the RI test. RI shows to be positive in the RI test and singly positive (HTG-A9) or doubly negative (HTG-A1, B6, and C3) in the duplication test. TD (targeted deletion) shows to be doubly negative in the duplication test and negative in the RI test (HTG-B8, B9, C1, and C4). HTG786 is a TD clone with 5′ SEXA1 / Syn 3’. HTG1047 is a TD clone with 5′ Syn / SEXA1 3’. (C) The allele sequence test for the clones derived from the donor plasmid with HPRT(ISCEI (545)Ex2Syn): The results from the allele sequence test for the HTG clones, which are doubly positive in the duplication test, are summarized as SEXA1 homo (5′ SEXA1 / SEXA1 3′), hetero (5′ Syn / SEXA1 3′ or 5′ SEXA1 / Syn 3′), and Syn homo (5′ Syn / Syn 3′). The results from the allele sequence test are summarized in .

Article Snippet: ISCEI site-linearized donor DNA: Each of the three types of ISCEI recognition site-containing designed donor plasmid DNA was digested with 1 unit of the I-SceI enzyme (NEB) per μg of DNA for 2 h at 37 °C and purified using an adsorption column (QIAGEN), followed by dissolution in endotoxin-free TE.

Techniques: Sequencing, Plasmid Preparation, Transfection, Electroporation, Clone Assay, Expressing, Derivative Assay

Journal: Frontiers in Genome Editing

Article Title: The precision strategy of human genome correction via a set of circular donor DNA and its cleaver

doi: 10.3389/fgeed.2026.1718252

Figure Lengend Snippet: InCDC is required for efficient targeted duplication. (A–C) A circular donor plasmid, pMB1KmHygTK-HPRT(ISCEI(318)Ex2Syn) (A) , pMB1KmHygTK-HPRT(ISCEI (545)Ex2Syn) (B) , or pMB1KmHygTK-HPRT(Ex2Syn (1195)ISCEI) (C) , along with plasmid DNA expressing the intra-cellular cleavage enzyme nls-I-SceI, was electroporated into the total number of cells transfected indicated in the first column. Alternatively, donor DNA linearized at the ISCEI site alone was electroporated into the total number of cells indicated in the first column. In the next five columns, the numbers show the viable cells transfected, which were calculated by multiplying the number of total cells plated by CFU (colony forming units)/500 total cell on two 9-cm diameter dishes; the overall integration clones as Hyg R colonies generated; the overall integration frequency as the fraction of the Hyg R colonies generated relative to the viable cells transfected; and the targeted duplication clones and the abortion-type clones in brackets, which were doubly positive and singly positive in the duplication test for Hyg R Tg R colonies, respectively. In the last three columns, the frequency of the targeted duplication clones per overall integration (Hyg R ) clone, the numbers of the Syn -retaining targeted duplication clones, and the frequency of the Syn -retaining targeted duplication clones per targeted duplication clone are shown. In these experiments, an advanced DMEM-based culture medium was used to obtain a more corrected value of transfected viable cells as the clonal capacity of the advanced DMEM-based culture medium is greater than that of the DMEM-based culture medium ( Materials and Methods ). The calculations using the Student T-TEST ( Excel ) and CHISQ.TEST ( Excel ) in (A–C) are shown in . The results from the allele sequence test in (A–C) are summarized in . (D) In the “InCDC” table (upper) and “Linearization” table (lower), lanes i and vi show the number of Hyg R clones and Hyg R Tg R clones generated, respectively; lanes ii and vii show the number of Hyg R Tg R clones classified into three types using the duplication test ; lanes iii and viii show each of the Hyg R Tg R clones also classified into two types using the RI test ; lanes iv and ix show the number of each of the forms [TD or non-TD (abortion-type TI and abortion-type RI, TD, or RI] of Hyg R Tg R clones classified, determined based on the two tests above; and lanes v and x show the non-TD frequency per overall integration (Hyg R ) clone. The calculations using the CHISQ.TEST ( Excel ) in panel (D) are shown in .

Article Snippet: ISCEI site-linearized donor DNA: Each of the three types of ISCEI recognition site-containing designed donor plasmid DNA was digested with 1 unit of the I-SceI enzyme (NEB) per μg of DNA for 2 h at 37 °C and purified using an adsorption column (QIAGEN), followed by dissolution in endotoxin-free TE.

Techniques: Plasmid Preparation, Expressing, Transfection, Clone Assay, Generated, Sequencing

Journal: Frontiers in Genome Editing

Article Title: The precision strategy of human genome correction via a set of circular donor DNA and its cleaver

doi: 10.3389/fgeed.2026.1718252

Figure Lengend Snippet: The assay system for human genome correction through intra-cellular circular donor cleavage-mediated targeted duplication and natural replacement. (A) The hypoxanthine phosphoribosyltransferase 1 ( HPRT1 ) gene is located between the PHF6 gene and the RPL36AP54 microRNA on the X chromosome in humans. It consists of nine exons and eight introns. The 5.4-kb section spanning intron 1, exon 2, intron 2, exon 3, and intron 3 was used as the targeting DNA, which is a probe used to search for homology across the entire chromosome. This 5.4-kb DNA is cloned into the pMB1KmHygTK plasmid vector ( Materials and Methods ), which has the bacterial replication origin (pMB1), the bacterial kanamycin-resistance gene ( Km ), and the fusion gene of the mammalian Hyg (hygromycin-resistant) gene with the HSV1tk (ganciclovir-sensitive) gene ( HygTK ). The I-SceI meganuclease-recognition site ISCEI and the designed sequence Syn are introduced into intron 1 and exon 2 of the cloned 5.4-kb DNA, respectively ( Materials and Methods ). SEXA1 , labeled on exon 2 of the targeted locus, indicates the wild-type allele ( SEXA1 allele), which is replaced with the Syn sequence ( Syn allele) in this assay system. The circular donor plasmid DNA consists of vector DNA (pink line) and homologous DNA (blue line). (B) A Syn -designed donor plasmid has synonymous codons (green letters) close to the SEXA1 site and is transfected to human cells with an artificial intra-cellular cleaver gene nls-I-SceI expression vector. The ISCEI sequence (red letters) of the Syn -designed donor plasmid is cleaved by the nls-I-SceI endonuclease. (C) Targeted duplication (TD) pathway: Homologous recombination between a Syn -designed donor plasmid and its homologous region around the exon 2 (TD1) produces a targeted duplication clone (TD2), which has a duplication structure with the Syn sequence and the SEXA1 sequence ( Syn / SEXA1 allele). The targeted duplication structure is naturally resolved through DNA replication during the S phase to form a single structure containing either the Syn allele (bold in TD3-1) or the SEXA1 allele (bold in TD3-2). Random integration (RI) pathway: Non-homologous recombination between the Syn -designed donor plasmid and a random site (RI1) produces a random integration clone (RI2). (D) The circular Syn -designed donor plasmid accesses the targeted counter chromosomal region (upper) to form the Syn -retaining targeted duplication structure, Syn / SEXA1 (center), which is confirmed by a couple of PCR-based tests as follows: (1) the duplication test is the PCR with the target-5′-outside forward primer (closed red arrow) and the plasmid-3′-inside reverse primer (closed violet arrow) and the other PCR with the plasmid-5′-inside forward primer (open violet arrow) and the target-3′-outside reverse primer (open red arrow); (2) the allele sequence test is the PCR with the Ex2–5′-outside primer and the Ex2–3′-outside primer (closed orange arrows), followed by its sequencing to verify the doublet chart of Syn and SEXA1 allele sequences (lower). (E) The natural replacement structure containing the Syn allele or the SEXA1 allele (center) is formed from a typical-targeted duplication structure (upper) by the popping-out of the circular DNA, which is confirmed using the following three tests: (1) the pop-out test is the PCR using target-5′-outside forward primer 2 (closed blue arrow) and target-3′-outside reverse primer 2 (open blue arrow) to verify the size characteristic of a natural replacement structure (the original size); (2) the Hyg test is the PCR with the 5′- Hyg forward primer and the 3′- Hyg reverse primer (closed green arrows) to verify the absence of the Hyg gene because the circular DNA that has popped out is removed by degradation or segregation in natural replacement cells; (3) the allele sequence test is the PCR with the Ex2–5′-outside forward primer and the Ex2–3′-outside reverse primer (closed orange arrows), followed by its sequencing to determine the sequence of the allele of a replacement clone, as shown in either a singlet chart of the Syn or SEXA1 allele sequence (lower).

Article Snippet: ISCEI site-linearized donor DNA: Each of the three types of ISCEI recognition site-containing designed donor plasmid DNA was digested with 1 unit of the I-SceI enzyme (NEB) per μg of DNA for 2 h at 37 °C and purified using an adsorption column (QIAGEN), followed by dissolution in endotoxin-free TE.

Techniques: Clone Assay, Plasmid Preparation, Sequencing, Labeling, Transfection, Expressing, Homologous Recombination

Journal: Frontiers in Genome Editing

Article Title: The precision strategy of human genome correction via a set of circular donor DNA and its cleaver

doi: 10.3389/fgeed.2026.1718252

Figure Lengend Snippet: Structures of the Syn allele and the SEXA1 allele in targeted duplication. (A) The Syn position of TD clones was verified by a couple of PCR-based tests as follows: (1) The 3.0-kb PCR with target-5′-outside forward primer 2 (closed blue arrow) and Syn reverse primer (opened green arrow: 5′-GCA​AAA​GAG​GTC​GAG​ATC​GTA​GCC-3′); (2) the 3.1-kb PCR with Syn forward primer (closed green arrow: 5′-GAA​CCA​GGC​TAC​GAT​CTC​GAC​CTC-3′) and target-3′-outside forward primer 2 (opened blue arrow). The 5′ Syn / SEXA1 3′ form leads to only the PCR product with the Syn reverse primer (upper). The 5′ SEXA1 / Syn 3′ form leads to only the PCR product with the Syn forward primer (middle). The 5′ Syn / Syn 3′ form leads to both types of PCR products (bottom). (B) The PCR products obtained from the above two tests for HTG-A1–A12, HTG-B1–B9, and HTG-C1∼C10 are shown by electrophoresis, which shows that 15, 5, 2, and 1 out of 23 TD clones were 5′ SEXA1 / SEXA1 3′, 5′ Syn / SEXA1 3′, 5′ SEXA1 / Syn 3′, and 5′ Syn / Syn 3′, respectively. HTG786 is a TD clone with 5′ SEXA1 / Syn 3’. HTG1047 is a TD clone with 5′ Syn / SEXA1 3’. (C–F) Crossover-type homologous recombination reactions produce four types of TD products: 5′ SEXA1 / SEXA1 3′, 5′ Syn / SEXA1 3′, 5′ SEXA1 / Syn 3′, and 5′ Syn / Syn 3’. Their reactions are explained as follows: 1. a donor plasmid DNA with a designed sequence (green) is cleaved at the ISCEI site within cells. 2. The double-strand break ends are resected by a putative exonuclease to form the double-strand gap. 3. The double-strand gap ends receive 5′-end resections to form the 3′-single-strand tails. 4. A 3′-single-strand tail is invaded into the homologous duplex of the chromosome to form the D-loop intermediate. 5. The D-loop intermediate is converted through strand exchange into the primed Holliday junction. 6. The repair synthesis starts at the 3′-end of the strand, which is stabilized in a structure of the primed Holliday junction to form the double Holliday junction. 7. The primed Holliday junction is horizontally resolved to form the single Holliday junction. 8. The single Holliday junction is vertically resolved to form the doublet structure comprising the donor DNA (green) and the chromosomal target DNA (blue), which possesses the donor allele duplex and the target allele duplex. Second on (C,D): A putative exonuclease degrades but remains the designed sequence of the donor DNA and forms the double-strand gap, which leads to the 5′ Syn / SEXA1 3′ doublet structure. However, when a putative exonuclease degrades beyond the designed sequence of the donor DNA and forms the longer double-strand gap, it leads to the 5′ SEXA1 / SEXA1 3′ doublet structure. Third on (E): When the double-strand gap ends receive the 5′-end resections associated with longer processivity to form the longer 3′-single-strand tail, it leads to the doublet structure with the single mismatch (seventh and eighth on (E) ) and produces the 5′ SEXA1 / Syn 3′ doublet structure after a DNA replication fork passes or the mismatch is repaired (ninth on (E) ). Fifth on (F): When the D-loop intermediate is converted through the branch migration associated with longer processivity to form another type of primed Holliday junction beyond the designed sequence, it leads to the doublet structure with the double mismatch (seventh and eighth on (F) ) and produces the 5′ Syn / Syn 3′ doublet structure after the double mismatch is repaired (ninth on (F) ).

Article Snippet: ISCEI site-linearized donor DNA: Each of the three types of ISCEI recognition site-containing designed donor plasmid DNA was digested with 1 unit of the I-SceI enzyme (NEB) per μg of DNA for 2 h at 37 °C and purified using an adsorption column (QIAGEN), followed by dissolution in endotoxin-free TE.

Techniques: Clone Assay, Electrophoresis, Homologous Recombination, Plasmid Preparation, Sequencing, Migration

Journal: Frontiers in Genome Editing

Article Title: The precision strategy of human genome correction via a set of circular donor DNA and its cleaver

doi: 10.3389/fgeed.2026.1718252

Figure Lengend Snippet: Messenger RNA molecules expressing intra-cellular cleaver nls-I-SceI lead to targeted duplication. (A) A circular donor plasmid, pMB1KmHygTK-HPRT(ISCEI(545)Ex2Syn), along with nls-I-SceI -expressing messenger RNA or the plasmid DNA expressing nls-I-SceI enzyme, shown in the first column, was transfected by electroporation into the number of total cells transfected, shown in the second column. In the next five columns, the numbers show the viable cells transfected, which were calculated by multiplying the number of total cells plated by the CFU (colony forming units)/500 total cells on two 9-cm-diameter dishes; the Hyg R colonies generated; the overall integration frequency as the fraction of the Hyg R colonies generated relative to the viable cells transfected; and the targeted duplication clones and the abortion-type clones in brackets, which are doubly positive and singly positive in the duplication test for Hyg R Tg R colonies, respectively. In the last three columns, the frequency of the targeted duplication clones per overall integration (Hyg R ) clone, the number of the Syn -retaining targeted duplication clones, and the frequency of the Syn -retaining targeted duplication clones per targeted duplication clone are shown. In these experiments, an advanced DMEM-based culture medium was used ( Materials and Methods ). The results from the allele sequence test are summarized in . (B) A circular donor plasmid, pMB1KmHygTK-ISCEI(545)Syn, was transfected by electroporation into the HT1080 cells infected with the nls-I-SceI -expressing Sendai virus (SeV) vector, SeV- nls-I-SceI , which is prepared by infecting the SeV vector, amplifying at 32 °C for 1 day, and storing at −80 °C ( Materials and Methods ). Before electroporation, the stock of HT1080 cells with SeV- nls-I-SceI was thawed and incubated at 37 °C for indicated days, as shown in the first column. In the next three columns, the numbers show the total cells transfected, the Hyg R colonies generated, and the targeted duplication clones and the abortion-type clones in brackets, which were doubly positive and singly positive in the duplication test for Hyg R Tg R colonies, respectively. In the last three columns, the frequency of the targeted duplication clones per overall integration (Hyg R ) clone, the number of the Syn -retaining targeted duplication clones, and the frequency of the Syn -retaining targeted duplication clones per targeted duplication clone are shown. In these experiments, a DMEM-based culture medium was used ( Materials and Methods ). The results from the allele sequence test are summarized in .

Article Snippet: ISCEI site-linearized donor DNA: Each of the three types of ISCEI recognition site-containing designed donor plasmid DNA was digested with 1 unit of the I-SceI enzyme (NEB) per μg of DNA for 2 h at 37 °C and purified using an adsorption column (QIAGEN), followed by dissolution in endotoxin-free TE.

Techniques: Expressing, Plasmid Preparation, Transfection, Electroporation, Generated, Clone Assay, Sequencing, Infection, Virus, Incubation

Journal: Frontiers in Genome Editing

Article Title: The precision strategy of human genome correction via a set of circular donor DNA and its cleaver

doi: 10.3389/fgeed.2026.1718252

Figure Lengend Snippet: Verification of the generality of PCR screening methods to isolate the targeted duplication clones out of overall integration clones. (A) Upper panel: A circular donor plasmid, pMB1KmHygTK-HPRT(ISCEI(545)Ex2Syn) was transfected to convert the SEXA1 allele of HT1080 to the Syn allele through intra-cellular circular donor cleavage (InCDC)-mediated targeted duplication (TD) and natural replacement. In line i, the sequence of the target allele ( SEXA1 ) in the HT1080 cell line is shown, which has original codons (blue letters) in exon 2 of the HPRT gene. The bold upper-lined letters are the SEXA1 site. In line ii, the sequence of the designed allele ( Syn ) is shown, which has synonymous codons (green letters) overlapped with the SEXA1 site. Lower panel [Targeted duplication (TD) screening of HT1080]: In lane iii, in the first three columns, the numbers show the total cells transfected by electroporation and the hygromycin-resistant (Hyg R ) colonies generated, and the TD dishes identified by PCR analysis for the bulk of the Hyg R colonies on every dish of the 16 dishes are shown. In the next two columns, the ID of the TD dishes identified and the presence or absence of the Syn allele in each of the TD dishes are shown. In the last two columns, the numbers of Syn -TD wells/TD wells/cell-growing wells in three 96-well plates [upper: feeder (HT1080 cell line) wells; lower: feeder-free wells] and their ID of the Syn -retaining TD wells identified are shown. The viability of cells derived from a #9-dish stock in 96-well plates was not significantly improved by the coexistence with feeder cells ( Materials and Methods ). (B) Upper panel: The Syn -retaining TD dishes, which contain targeted duplication clones with Syn allele sequences, were screened using two PCR-based assays as follows: the TD-screening PCR with the plasmid-5′-inside forward primer (opened violet arrow) and the target-3′-outside reverse primer (opened blue arrow); the Syn -retaining TD-screening PCR with the target-5′-outside forward primer (closed blue arrow) and the Syn reverse primer (opened green arrow). Lower left panel: The circular donor plasmid, pMB1KmHygTK-HPRT(ISCEI(545)Ex2Syn), was transfected, and the resultant 16 dishes were screened by TD screening PCR and Syn -TD screening PCR, with both PCR products electrophoresed, leading to the identification of Syn -retaining TD dish #9. Lower right panel: Six wells of 96-well plates were identified as Syn -TD clones derived from #9-dish stock by TD screening PCR and Syn -TD screening PCR, with both PCR products electrophoresed, leading to the identification of six Syn -retaining TD wells. HTG-A4 is a TD clone with 5′ Syn / SEXA1 3’. (C) A TD structure with Syn / SEXA1 hetero-allele sequences forms one of the two types of natural replacement structures: with the Syn allele (left) or with the SEXA1 allele (right), which was verified using three PCR-based tests: (1) pop-out test (closed blue arrow and open blue arrow) to verify a natural replacement structure; (2) Hyg test (green arrows) to verify the popping out of the circular plasmid DNA; and (3) allele sequence test (orange arrows) to determine either allele of a replacement clone. One TD well-clone d9p5F12 is shown in the first column. In the following five columns, the numbers show the obtained GCV R colonies, Syn allele clones, SEXA1 allele clones, and TK-deficient TD clones, in which duplication tests were still positive , and others [TD-derived deletion clones, in which all of pop-out test, Hyg test, and duplication test were negative ]. The complete results of the Syn allele or the SEXA1 allele of pop-out type GCV clones are summarized in . The complete results of the HygTK sequence of TK-deficient TD-type GCV clones are summarized in . Upper panel: The sequence data from the Syn clones identified using the allele sequence test among HTG786-derived pop-out type GCV R clones (lanes i and ii in ) are shown, each of which was the singlet chart of the Syn allele sequence. Lower panel: The sequence data from the Syn clones identified using the allele sequence test among d9p5F12 -derived pop-out-type GCV R clones (lanes i and ii in ) are shown, each of which was the singlet chart of the Syn allele sequence. The sequence data from their SEXA1 clones are also shown in .

Article Snippet: ISCEI site-linearized donor DNA: Each of the three types of ISCEI recognition site-containing designed donor plasmid DNA was digested with 1 unit of the I-SceI enzyme (NEB) per μg of DNA for 2 h at 37 °C and purified using an adsorption column (QIAGEN), followed by dissolution in endotoxin-free TE.

Techniques: Clone Assay, Plasmid Preparation, Transfection, Sequencing, Electroporation, Generated, Derivative Assay

Journal: Frontiers in Genome Editing

Article Title: The precision strategy of human genome correction via a set of circular donor DNA and its cleaver

doi: 10.3389/fgeed.2026.1718252

Figure Lengend Snippet: Several hundred base pairs in distance between the cleavage site and the designed sequence on a circular donor plasmid are required to retain the designed allele. (A) A circular donor plasmid, pMB1KmHygTK-HPRT(ISCEI(318)Ex2Syn), pMB1KmHygTK-HPRT(ISCEI(545)Ex2Syn), or pMB1KmHygTK-HPRT(Ex2Syn(1195)ISCEI), consists of the vector DNA (pink line) and the homologous DNA (blue line) with the Syn sequence and the ISCEI cleavage site. In the left column, three types of circular donor plasmids depict the length of left homology, the distance between the cleavage point and the Syn sequence, and the length of right homology with a cleavage type (no cleavage, intra-cellular cleavage, or linearized at the ISCEI site). In the following three columns, the numbers show the total cells transfected by electroporation, the whole integration clones observed as hygromycin-resistant (Hyg R ) colonies, the TD clones, and the abortion clones in brackets, which were doubly positive and singly positive in the duplication test , respectively. In the last three columns, the frequency of the TD clones relative to the whole Hyg R colonies, the number of the Syn -retaining TD clones, and the frequency of the Syn -retaining TD clones relative to the TD clones are shown. In the case of the intra-cellular cleavage type, the nls-I-SceI expression plasmid, pCI- nls-I-SceI or pCAG.I-SceI plasmid (bottom on lane v), was co-transfected with a circular donor DNA ( Materials and Methods ). The calculations by CHISQ.TEST ( Excel ) are shown in . (B) The duplication test for the clones derived from the donor plasmid with HPRT(ISCEI(545)Ex2Syn): the results from the duplication test for 12 hygromycin-resistant and 6-thioguanine-resistant (Hyg R TG R ) clones (HTG-A1–A12) out of 1,768 Hyg R clones (top of lane iii in panel (A) ) are shown. Out of 12, 10 clones are doubly positive, 1 clone (HTG-A9) is singly positive (Ab: Abortion), and 1 clone (HTG-A1) is doubly negative in the duplication test. The results for 9 Hyg R TG R clones (HTG-B1–B9) out of 1708 Hyg R clones [Center of lane iii in (A) ] are shown. Out of 9, 6 clones are doubly positive and 3 clones (HTG-B5, B8, and B9) are doubly negative in the test. The results for 10 Hyg R TG R clones (HTG-C1–C10) out of 2,872 Hyg R clones (bottom of lane iii in panel A) are shown. Out of 10, 7 clones are doubly positive and 3 clones (HTG-C1, C3, and C4) are doubly negative in the test. Random integration (RI) test for the clones derived from the donor plasmid with HPRT(ISCEI(545)Ex2Syn): The results from the RI test for HTG-A1–A12, HTG-B1–B9, and HTG-C1–C10 clones are shown, based on the PCR assay with target-5′-outside forward primer 2 (closed blue arrow) and target-3′-outside reverse primer 2 (open blue arrow), which is the same primer pair as that in the pop-out test . TD shows to be doubly positive in the duplication test and negative in the RI test. RI shows to be positive in the RI test and singly positive (HTG-A9) or doubly negative (HTG-A1, B6, and C3) in the duplication test. TD (targeted deletion) shows to be doubly negative in the duplication test and negative in the RI test (HTG-B8, B9, C1, and C4). HTG786 is a TD clone with 5′ SEXA1 / Syn 3’. HTG1047 is a TD clone with 5′ Syn / SEXA1 3’. (C) The allele sequence test for the clones derived from the donor plasmid with HPRT(ISCEI (545)Ex2Syn): The results from the allele sequence test for the HTG clones, which are doubly positive in the duplication test, are summarized as SEXA1 homo (5′ SEXA1 / SEXA1 3′), hetero (5′ Syn / SEXA1 3′ or 5′ SEXA1 / Syn 3′), and Syn homo (5′ Syn / Syn 3′). The results from the allele sequence test are summarized in .

Article Snippet: ISCEI site-linearized donor DNA: Each of the three types of ISCEI recognition site-containing designed donor plasmid DNA was digested with 1 unit of the I-SceI enzyme (NEB) per μg of DNA for 2 h at 37 °C and purified using an adsorption column (QIAGEN), followed by dissolution in endotoxin-free TE.

Techniques: Sequencing, Plasmid Preparation, Transfection, Electroporation, Clone Assay, Expressing, Derivative Assay

Journal: Frontiers in Genome Editing

Article Title: The precision strategy of human genome correction via a set of circular donor DNA and its cleaver

doi: 10.3389/fgeed.2026.1718252

Figure Lengend Snippet: InCDC is required for efficient targeted duplication. (A–C) A circular donor plasmid, pMB1KmHygTK-HPRT(ISCEI(318)Ex2Syn) (A) , pMB1KmHygTK-HPRT(ISCEI (545)Ex2Syn) (B) , or pMB1KmHygTK-HPRT(Ex2Syn (1195)ISCEI) (C) , along with plasmid DNA expressing the intra-cellular cleavage enzyme nls-I-SceI, was electroporated into the total number of cells transfected indicated in the first column. Alternatively, donor DNA linearized at the ISCEI site alone was electroporated into the total number of cells indicated in the first column. In the next five columns, the numbers show the viable cells transfected, which were calculated by multiplying the number of total cells plated by CFU (colony forming units)/500 total cell on two 9-cm diameter dishes; the overall integration clones as Hyg R colonies generated; the overall integration frequency as the fraction of the Hyg R colonies generated relative to the viable cells transfected; and the targeted duplication clones and the abortion-type clones in brackets, which were doubly positive and singly positive in the duplication test for Hyg R Tg R colonies, respectively. In the last three columns, the frequency of the targeted duplication clones per overall integration (Hyg R ) clone, the numbers of the Syn -retaining targeted duplication clones, and the frequency of the Syn -retaining targeted duplication clones per targeted duplication clone are shown. In these experiments, an advanced DMEM-based culture medium was used to obtain a more corrected value of transfected viable cells as the clonal capacity of the advanced DMEM-based culture medium is greater than that of the DMEM-based culture medium ( Materials and Methods ). The calculations using the Student T-TEST ( Excel ) and CHISQ.TEST ( Excel ) in (A–C) are shown in . The results from the allele sequence test in (A–C) are summarized in . (D) In the “InCDC” table (upper) and “Linearization” table (lower), lanes i and vi show the number of Hyg R clones and Hyg R Tg R clones generated, respectively; lanes ii and vii show the number of Hyg R Tg R clones classified into three types using the duplication test ; lanes iii and viii show each of the Hyg R Tg R clones also classified into two types using the RI test ; lanes iv and ix show the number of each of the forms [TD or non-TD (abortion-type TI and abortion-type RI, TD, or RI] of Hyg R Tg R clones classified, determined based on the two tests above; and lanes v and x show the non-TD frequency per overall integration (Hyg R ) clone. The calculations using the CHISQ.TEST ( Excel ) in panel (D) are shown in .

Article Snippet: ISCEI site-linearized donor DNA: Each of the three types of ISCEI recognition site-containing designed donor plasmid DNA was digested with 1 unit of the I-SceI enzyme (NEB) per μg of DNA for 2 h at 37 °C and purified using an adsorption column (QIAGEN), followed by dissolution in endotoxin-free TE.

Techniques: Plasmid Preparation, Expressing, Transfection, Clone Assay, Generated, Sequencing

Journal: Frontiers in Genome Editing

Article Title: The precision strategy of human genome correction via a set of circular donor DNA and its cleaver

doi: 10.3389/fgeed.2026.1718252

Figure Lengend Snippet: The assay system for human genome correction through intra-cellular circular donor cleavage-mediated targeted duplication and natural replacement. (A) The hypoxanthine phosphoribosyltransferase 1 ( HPRT1 ) gene is located between the PHF6 gene and the RPL36AP54 microRNA on the X chromosome in humans. It consists of nine exons and eight introns. The 5.4-kb section spanning intron 1, exon 2, intron 2, exon 3, and intron 3 was used as the targeting DNA, which is a probe used to search for homology across the entire chromosome. This 5.4-kb DNA is cloned into the pMB1KmHygTK plasmid vector ( Materials and Methods ), which has the bacterial replication origin (pMB1), the bacterial kanamycin-resistance gene ( Km ), and the fusion gene of the mammalian Hyg (hygromycin-resistant) gene with the HSV1tk (ganciclovir-sensitive) gene ( HygTK ). The I-SceI meganuclease-recognition site ISCEI and the designed sequence Syn are introduced into intron 1 and exon 2 of the cloned 5.4-kb DNA, respectively ( Materials and Methods ). SEXA1 , labeled on exon 2 of the targeted locus, indicates the wild-type allele ( SEXA1 allele), which is replaced with the Syn sequence ( Syn allele) in this assay system. The circular donor plasmid DNA consists of vector DNA (pink line) and homologous DNA (blue line). (B) A Syn -designed donor plasmid has synonymous codons (green letters) close to the SEXA1 site and is transfected to human cells with an artificial intra-cellular cleaver gene nls-I-SceI expression vector. The ISCEI sequence (red letters) of the Syn -designed donor plasmid is cleaved by the nls-I-SceI endonuclease. (C) Targeted duplication (TD) pathway: Homologous recombination between a Syn -designed donor plasmid and its homologous region around the exon 2 (TD1) produces a targeted duplication clone (TD2), which has a duplication structure with the Syn sequence and the SEXA1 sequence ( Syn / SEXA1 allele). The targeted duplication structure is naturally resolved through DNA replication during the S phase to form a single structure containing either the Syn allele (bold in TD3-1) or the SEXA1 allele (bold in TD3-2). Random integration (RI) pathway: Non-homologous recombination between the Syn -designed donor plasmid and a random site (RI1) produces a random integration clone (RI2). (D) The circular Syn -designed donor plasmid accesses the targeted counter chromosomal region (upper) to form the Syn -retaining targeted duplication structure, Syn / SEXA1 (center), which is confirmed by a couple of PCR-based tests as follows: (1) the duplication test is the PCR with the target-5′-outside forward primer (closed red arrow) and the plasmid-3′-inside reverse primer (closed violet arrow) and the other PCR with the plasmid-5′-inside forward primer (open violet arrow) and the target-3′-outside reverse primer (open red arrow); (2) the allele sequence test is the PCR with the Ex2–5′-outside primer and the Ex2–3′-outside primer (closed orange arrows), followed by its sequencing to verify the doublet chart of Syn and SEXA1 allele sequences (lower). (E) The natural replacement structure containing the Syn allele or the SEXA1 allele (center) is formed from a typical-targeted duplication structure (upper) by the popping-out of the circular DNA, which is confirmed using the following three tests: (1) the pop-out test is the PCR using target-5′-outside forward primer 2 (closed blue arrow) and target-3′-outside reverse primer 2 (open blue arrow) to verify the size characteristic of a natural replacement structure (the original size); (2) the Hyg test is the PCR with the 5′- Hyg forward primer and the 3′- Hyg reverse primer (closed green arrows) to verify the absence of the Hyg gene because the circular DNA that has popped out is removed by degradation or segregation in natural replacement cells; (3) the allele sequence test is the PCR with the Ex2–5′-outside forward primer and the Ex2–3′-outside reverse primer (closed orange arrows), followed by its sequencing to determine the sequence of the allele of a replacement clone, as shown in either a singlet chart of the Syn or SEXA1 allele sequence (lower).

Article Snippet: ISCEI site-linearized donor DNA: Each of the three types of ISCEI recognition site-containing designed donor plasmid DNA was digested with 1 unit of the I-SceI enzyme (NEB) per μg of DNA for 2 h at 37 °C and purified using an adsorption column (QIAGEN), followed by dissolution in endotoxin-free TE.

Techniques: Clone Assay, Plasmid Preparation, Sequencing, Labeling, Transfection, Expressing, Homologous Recombination

Journal: Frontiers in Genome Editing

Article Title: The precision strategy of human genome correction via a set of circular donor DNA and its cleaver

doi: 10.3389/fgeed.2026.1718252

Figure Lengend Snippet: Messenger RNA molecules expressing intra-cellular cleaver nls-I-SceI lead to targeted duplication. (A) A circular donor plasmid, pMB1KmHygTK-HPRT(ISCEI(545)Ex2Syn), along with nls-I-SceI -expressing messenger RNA or the plasmid DNA expressing nls-I-SceI enzyme, shown in the first column, was transfected by electroporation into the number of total cells transfected, shown in the second column. In the next five columns, the numbers show the viable cells transfected, which were calculated by multiplying the number of total cells plated by the CFU (colony forming units)/500 total cells on two 9-cm-diameter dishes; the Hyg R colonies generated; the overall integration frequency as the fraction of the Hyg R colonies generated relative to the viable cells transfected; and the targeted duplication clones and the abortion-type clones in brackets, which are doubly positive and singly positive in the duplication test for Hyg R Tg R colonies, respectively. In the last three columns, the frequency of the targeted duplication clones per overall integration (Hyg R ) clone, the number of the Syn -retaining targeted duplication clones, and the frequency of the Syn -retaining targeted duplication clones per targeted duplication clone are shown. In these experiments, an advanced DMEM-based culture medium was used ( Materials and Methods ). The results from the allele sequence test are summarized in . (B) A circular donor plasmid, pMB1KmHygTK-ISCEI(545)Syn, was transfected by electroporation into the HT1080 cells infected with the nls-I-SceI -expressing Sendai virus (SeV) vector, SeV- nls-I-SceI , which is prepared by infecting the SeV vector, amplifying at 32 °C for 1 day, and storing at −80 °C ( Materials and Methods ). Before electroporation, the stock of HT1080 cells with SeV- nls-I-SceI was thawed and incubated at 37 °C for indicated days, as shown in the first column. In the next three columns, the numbers show the total cells transfected, the Hyg R colonies generated, and the targeted duplication clones and the abortion-type clones in brackets, which were doubly positive and singly positive in the duplication test for Hyg R Tg R colonies, respectively. In the last three columns, the frequency of the targeted duplication clones per overall integration (Hyg R ) clone, the number of the Syn -retaining targeted duplication clones, and the frequency of the Syn -retaining targeted duplication clones per targeted duplication clone are shown. In these experiments, a DMEM-based culture medium was used ( Materials and Methods ). The results from the allele sequence test are summarized in .

Article Snippet: ISCEI site-linearized donor DNA: Each of the three types of ISCEI recognition site-containing designed donor plasmid DNA was digested with 1 unit of the I-SceI enzyme (NEB) per μg of DNA for 2 h at 37 °C and purified using an adsorption column (QIAGEN), followed by dissolution in endotoxin-free TE.

Techniques: Expressing, Plasmid Preparation, Transfection, Electroporation, Generated, Clone Assay, Sequencing, Infection, Virus, Incubation