mb2cas12a  (Addgene inc)

Journal: Frontiers in Genome Editing

Article Title: Hs1Cas12a and Ev1Cas12a confer efficient genome editing in plants

doi: 10.3389/fgeed.2023.1251903

Figure Lengend Snippet: Screening of novel Cas12a orthologs in rice protoplasts. (A) , phylogenetic tree of Cas12a nucleases using MEGA11. Amino acid sequences of Cas12a ortholog candidates was acquired from Joint Genome Institute Microbial Genomes and Microbiomes (JGI IMG/M) database by using LbCas12a, FnCas12a, ErCas12a (MAD7) and Mb2Cas12a as the BLAST queries. Seventeen novel Cas12a nuclease candidates were labeled with red dots. (B) , targeted mutagenesis efficiencies (percentage) of 17 novel Cas12a orthologs at four target sites with TTTV and VTTV PAMs in rice protoplasts. WT, protoplasts transformed with water. LbCas12a and Mb2Cas12a were used as controls. Data are presented as mean values ±SEM. n = 3 biologically independent samples.

Article Snippet: LbCas12a and Mb2Cas12a were used as controls. pYPQ141-ZmUbi-RZ-Lb (Addgene #86197, ) was used for crRNA cloning of LbCas12a.

Techniques: Labeling, Mutagenesis, Transformation Assay

Journal: Frontiers in Genome Editing

Article Title: Hs1Cas12a and Ev1Cas12a confer efficient genome editing in plants

doi: 10.3389/fgeed.2023.1251903

Figure Lengend Snippet: Genome editing of Ev1Cas12a and Hs1Cas12a in rice protoplasts at low temperatures. (A) , multiplexed genome editing of three crRNAs to target five sites using a dual ZmUbi promoter and tandem HH-crRNA-HDV system. (B) , total mutation and deletion efficiencies (percentage) of Ev1Cas12a and Hs1Cas12a at five target sites with TTTV and VTTV PAMs at 28°C, 25°C, and 22°C in rice protoplasts compared with LbCas12a and Mb2Cas12a. WT, protoplasts transformed with water. (C) , deletion size of Ev1Cas12a and Hs1Cas12a at CC1-TTTC site in rice protoplasts. (D) , deletion position of Ev1Cas12a and Hs1Cas12a at CC1-CTTC site in rice protoplasts. PAM sequence is highlighted in red and protospacer sequence is highlighted in green. Data are presented as mean values ±SEM. n = 3 biologically independent samples.

Article Snippet: LbCas12a and Mb2Cas12a were used as controls. pYPQ141-ZmUbi-RZ-Lb (Addgene #86197, ) was used for crRNA cloning of LbCas12a.

Techniques: Mutagenesis, Transformation Assay, Sequencing

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire.

doi: 10.1016/j.ymthe.2021.10.027

Figure Lengend Snippet: Figure 1. Targeting the conserved region of JH4 gene requires a Cas12a ortholog recognizing non-canonical PAMs (A) A representation of the coding region of an antibody heavy-chain variable region is presented. As indicated, the HCDR3 (green) is encoded by the 30 of a recombined V gene, a D gene, and the 50 of a J-chain. To insert a common HCDR3 into a diverse population of BCR, the gRNA of a CRISPR effector protein must complement a conserved HC region at the 30 end of the recombined J-gene, while cleaving a more variable region near the site of HCDR3 insertion. Note that, unlike Cas9, Cas12a cleaves distally from its PAM and seed regions. The preferred PAM recognition sequence of commonly studied Cas12a orthologs is TTTV. However, as shown, JH4, the most frequently used JH gene in all species, contains optimally located GTTC and TTCC PAM sequences, located 30 of the HCDR3-encoding sequence but oriented Cas12a cleavage within this sequence. This PAM, sequence of the gRNA, and the Cas12a cut sites are indicated. (B) To identify a Cas12a ortholog efficient at cleaving these non-canonical PAM motifs, the human B cell line Jeko-1 was co-transfected with two plasmids encoding the CRISPR protein (BsCas12a, TsCas12a, Mb2Cas12a, or Mb3Cas12a), and their corre- sponding gRNA. Control samples were transfected without gRNA plasmids. Targeting efficiency was measured by flow cytometry as loss of IgM expression. Among these Cas12a orthologs, Mb2Cas12 most efficiently cleaved the J-chain region initiated with GTTC and TTCC (orange). Error bars indicate standard error (SEM) of two independent experiments, and asterisks indicate statistical significance relative to controls. Statistical difference was determined by non-paired Student’s t-test (****p < 0.0001). (C) Mb2Cas12a RNP was compared with commercial AsCas12a RNP for their ability cleave four distinct regions in the HCDR3-encoding region of Jeko-1 cells. Loss of IgM expression indicates successful introduction of a double-strand break and NHEJ. RNP with gRNA targeting an irrelevant site (EMX1) was used as control. (D) Results of three experiments similar to that shown in (B). Error bars indicate SEM of at least two independent experiments. Asterisks indicate significant differences from the canonical TTTG PAM (Mb2Cas12a or AsCas12a, respectively). Statistical difference was determined by non-paired Student’s t-test (****p < 0.0001).

Article Snippet: Wild-type Mb2Cas12a (pcDNA3.1-hMb2Cpf1), Mb3Cas12a (pcDNA3.1-hMb3Cpf1), TsCas12a (pcDNA3.1-hTsCpf1), and BsCas12a (pcDNA3.1-hBsCpf1) plasmids were gifts from Dr. Feng Zhang (Addgene: 92,292, 92,293, 92,267, 92,300). pMAL-hisLbCpf1-EC was a gift from Dr. Jin-Soo Kim (Addgene: 79,008) and was used to express Mb2Cas12a in E. coli for protein production.

Techniques: CRISPR, Sequencing, Transfection, Control, Cytometry, Expressing

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire.

doi: 10.1016/j.ymthe.2021.10.027

Figure Lengend Snippet: Figure 2. Optimization of ssDNA templates for Mb2Cas12a-mediated editing the HCDR3-encoding region of a human B cell line (A) A diagram representing four HDRTs used in (B–E). Specifically, sense and anti-sense forms of HDRT-A, were used to replace a 9-nt region (gray) with a 39-nt insert (green), and both forms of HDRT-B were used to replace a 36-nt region with a 69-nt region; 50-nt homology arms of the sense and anti-sense forms are represented in red and blue, respectively. SpCas9 (cyan) and Mb2Cas12A (orange) cleavage sites of the target strand (complementary to gRNA) are indicated by arrows. Note that paired Cas9 and Cas12a cleavage sites are separated by at most 5 nts. (B) A representative example of an experiment used to generate (C–E) in which editing efficiency of Mb2Cas12A or SpCas9 RNP was monitored through recognition of an HA tag introduced into the HCDR3 of the Jeko-1 cell BCR by flow cytometry. Control cells were electroporated with Mb2Cas12a RNP without an HDRT. (C) A comparison of Mb2Cas12a (Mb2) and SpCas9 (Cas9) knock-in efficiencies, measured as described in (B), with the four different HDRTs and for all four sites shown in (A). Differences between Mb2 and Cas9, and among the four sites, are not significant (n.s.). The same data generated for (C) was replotted according to whether the sense or anti-sense HDRT was used (D), or whether the HDRT complemented the gRNA target or non-target strand. (E) Non-target strand is the PAM-containing strand, and the target strand is the strand annealed to gRNA. Again, as indicated, most differences were not significant. However, the HDRT- complementary to the Mb2Cas12a gRNA target strand were slightly more efficient than those complementary to the non-target strand (p = 0.027). Dots in (C–E) represent pooled data from two independent experiments. Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: Wild-type Mb2Cas12a (pcDNA3.1-hMb2Cpf1), Mb3Cas12a (pcDNA3.1-hMb3Cpf1), TsCas12a (pcDNA3.1-hTsCpf1), and BsCas12a (pcDNA3.1-hBsCpf1) plasmids were gifts from Dr. Feng Zhang (Addgene: 92,292, 92,293, 92,267, 92,300). pMAL-hisLbCpf1-EC was a gift from Dr. Jin-Soo Kim (Addgene: 79,008) and was used to express Mb2Cas12a in E. coli for protein production.

Techniques: Cytometry, Control, Comparison, Knock-In, Generated

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire.

doi: 10.1016/j.ymthe.2021.10.027

Figure Lengend Snippet: Figure 4. The BCR specificity of Jeko-1 cells can be reprogrammed with a novel HCDR3 (A) The amino acid sequence of the native Jeko-1 cell HCDR3 region and those of the HIV-1 neutralizing antibodies PG9 and PG16 are shown. The amino acid translations of the new sequences from the four HDRTs used in the subsequent panels are represented in green italics, in the context of the remaining Jeko-1 region. (B) Mb2Cas12a RNP targeting the GTTC PAM of site 4 in Jeko-1 cells shown in Figure 2B were co-electroporated with the indicated HDRT. Editing efficiency was monitored on the vertical axis by flow cytometry with fluorescently labeled PSG2, an antibody that recognizes sulfotyrosines within the PG9 and PG16 HCDR3 region, a similarly labeled HIV SOSIP or E2p. The horizontal axis indicates IgM expression, and its loss indicates imprecise NHEJ after Mb2Cas12a-mediated cleavage. Note that introduction of a PG16 HCDR3 was efficient, as indicated by PSG2 recognition, but unlike the PG9 HCDR3, it did not bind the Env trimer. Cells edited to express an HA tag did not bind any reagent. SOSIP proteins were derived from the BG505 HIV-1 isolate. (C) A summary of three independent experiments similar to that shown in (B) of flow cytometric studies used to generate (B). Error bars indicate SD. (D) Jeko-1 edited with PG9-CAR HDRT were enriched by FACS with the anti-sulfotyrosine antibody PSG2. (E) Cells enriched in (D) were analyzed 2 weeks later by flow cytometry for their ability to bind PSG2, a BG505-based nanoparticle (BG505-E2p), SOSIP trimers derived from the indicated HIV-1 isolate, or an V2 apex negative mutant (dBG505-SOSIP). Gray control indicates wild-type Jeko-1 cells. (F) Unedited Jeko-1 cells and those edited with PG9-CAR HDRT without sorting, or sorted with PSG2 or with E2p, were analyzed by NGS of the VDJ region. Sequences were divided into four categories, depending on whether the edited sequence exactly matched the HDRT (Perfect HDR), whether the HDRT sequence was visible but modified (Imperfect HDR), whether the original Jeko-1 HCDR3 region was intact (Original), or whether this region was modified by NHEJ as indicated by the presence of insertions or deletions (Indel). Representative examples of each category are shown below the charts.

Article Snippet: Wild-type Mb2Cas12a (pcDNA3.1-hMb2Cpf1), Mb3Cas12a (pcDNA3.1-hMb3Cpf1), TsCas12a (pcDNA3.1-hTsCpf1), and BsCas12a (pcDNA3.1-hBsCpf1) plasmids were gifts from Dr. Feng Zhang (Addgene: 92,292, 92,293, 92,267, 92,300). pMAL-hisLbCpf1-EC was a gift from Dr. Jin-Soo Kim (Addgene: 79,008) and was used to express Mb2Cas12a in E. coli for protein production.

Techniques: Sequencing, Cytometry, Labeling, Expressing, Derivative Assay, Mutagenesis, Control

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire.

doi: 10.1016/j.ymthe.2021.10.027

Figure Lengend Snippet: Figure 5. Editing primary human B cells with HDRT recognizing consensus sequences of multiple VH families (A) A panel of PG9-CAR HDRT with homology arms complementary to JH4 and to consensus VH1-, VH3-, and VH4-family sequences were evaluated for their ability to edit primary human B cells. Cells electroporated with Mb2Cas12a RNP and PG9- CAR HDRT were analyzed by flow cytometry with the anti-sulfotyrosine antibody PSG2 modified with two distinct fluorophores to eliminate non-specific binding from either fluorophore. (B) A summary of results from experiments similar to that shown in (A), using primary B cells from three independent donors. Note that a mixture of three HDRTs edited more cells than any individual HDRT. Null indicates that cells were not electroporated and control indicates cells electroporated with Mb2Cas12a RNP and an HDRT that is not homologous to any sequence in the human genome. Mix indicates cells electroporated with RNP and an equimolar mixture of HDRT with VH1-, VH3-, and VH4-specific homology arms. Error bars indicate range of three independent experiments, and asterisks indicate statistical significance calculated by one-way ANOVA with Tukey’s multiple comparison test (*p < 0.5; **p < 0.01; ****p < 0.0001). (C) NGS analysis of primary B cells from two human donors. %HDR was quantified as described in Figure 4F, including both perfect and imperfect. The portion of VH-family of edited cells in HDR-positive sequences was also counted.

Article Snippet: Wild-type Mb2Cas12a (pcDNA3.1-hMb2Cpf1), Mb3Cas12a (pcDNA3.1-hMb3Cpf1), TsCas12a (pcDNA3.1-hTsCpf1), and BsCas12a (pcDNA3.1-hBsCpf1) plasmids were gifts from Dr. Feng Zhang (Addgene: 92,292, 92,293, 92,267, 92,300). pMAL-hisLbCpf1-EC was a gift from Dr. Jin-Soo Kim (Addgene: 79,008) and was used to express Mb2Cas12a in E. coli for protein production.

Techniques: Cytometry, Binding Assay, Control, Sequencing, Comparison

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire.

doi: 10.1016/j.ymthe.2021.10.027

Figure Lengend Snippet: Figure 6. Reprogrammed primary human B cells retain VH-gene and light-chain diversity Primary cells were electroporated with Mb2Cas12a RNP and HDRT encoding an HA tag (A) or the HCDR3 regions of the HIV-1 neutralizing antibodies PG9 (B) and PG16 (C), with the same mixture of homology arms as those used in Figure 5. Cells were sorted with an anti-HA antibody (HA tag [A]) or an SOSIP trimer derived from the CRF_AG_250 isolate (B and C). Approximately 2% to 3% of cells were sorted by the SOSIP trimer. Edited cells were analyzed by NGS before and after sorting, the percentages of PG9 and PG16-HCDR3 inserts were enriched from 1% to 30%. The frequency of each VH1-, VH3-, and VH4-family genes from BCR sequences bearing the desired inserts was measured. Flow cytometry histograms display one of two experiments with similar results, and bar graphs indicate the mean of those two experiments. (D) The frequency of each light chain genes of PG9-HCR3 inserted primary cells pre (blue) and post (red) sorting was analyzed by NGS. Bar graphs indicate the mean of two independent experiments.

Article Snippet: Wild-type Mb2Cas12a (pcDNA3.1-hMb2Cpf1), Mb3Cas12a (pcDNA3.1-hMb3Cpf1), TsCas12a (pcDNA3.1-hTsCpf1), and BsCas12a (pcDNA3.1-hBsCpf1) plasmids were gifts from Dr. Feng Zhang (Addgene: 92,292, 92,293, 92,267, 92,300). pMAL-hisLbCpf1-EC was a gift from Dr. Jin-Soo Kim (Addgene: 79,008) and was used to express Mb2Cas12a in E. coli for protein production.

Techniques: Derivative Assay, Flow Cytometry