pypq284  (Addgene inc)

Journal: bioRxiv

Article Title: Efficient reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire

doi: 10.1101/2021.04.01.437943

Figure Lengend Snippet: ( 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 3’ of a recombined V gene, a D gene, and the 5’ of a J-chain. To insert a common HCDR3 into a diverse population of BCR, the guide RNA (gRNA) of a CRISPR effector protein must complement a conserved HC region at the 3’ 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 3’ of the HCDR3-encoding sequence but oriented Cas12a cleavage within the 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 transfected with plasmids encoding BsCas12a, TsCas12a, Mb2Cas12a, or Mb3Cas12a. 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 indicted range of two independent experiments, and asterisks indicate statistical significance relative to controls. Statistical difference were determined by non-paired Students t-test, (****, p<0.0001). ( C ) Mb2Cas12 RNP were 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 inexact NHEJ. ( D ) Results of three experiments similar to that shown in panel C. Error bars indicate standard error (SEM). Asterisks indicted significant differences from the canonical TTTG PAM (Mb2Cas12a-RNP or AsCas12a, respectively). Statistical difference were determined by non-paired Students 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 plasmid numbers 69982, 69988, 69983, and 42230, respectively). pMAL-his-LbCpf1-EC was a gift from Dr. Jin-Soo Kim (Addgene plasmid number 79008) and was used to express Mb2Cas12a in E. coli for protein production.

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

Journal: bioRxiv

Article Title: Efficient reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire

doi: 10.1101/2021.04.01.437943

Figure Lengend Snippet: ( A ) A diagram representing four HDR templates (HDRT) used in panels B-E. Specifically, sense and anti-sense forms of HDRT-A, were used to replace a 9-nucleotide (nt) region (grey) with 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 antisense forms are represented in red and blue, respectively. SpCas9 (cyan) and Mb2Cas12 (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 five nucleotides. ( B ) A representative example of an experiment used to generate panels C-E in which editing efficiency of MbCas12A or SpCas9 RNP is 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 panel B, for all four sites shown in panel A. Differences between Mb2 and Cas9, and among the four sites, are not significant (n.s.). The same data generated for panel C was replotted according to whether the sense or anti-sense HDRT were 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 plasmid numbers 69982, 69988, 69983, and 42230, respectively). pMAL-his-LbCpf1-EC was a gift from Dr. Jin-Soo Kim (Addgene plasmid number 79008) and was used to express Mb2Cas12a in E. coli for protein production.

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

Journal: bioRxiv

Article Title: Efficient reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire

doi: 10.1101/2021.04.01.437943

Figure Lengend Snippet: ( A ) A model showing where a 3’ mismatch tail occurs. A cut site (yellow) is introduced into a region of the gene targeted for replacement (grey), asymmetrically dividing this region. Efficient 5’ to 3’ resection exposes two 3’ ends. An HDRT can complement a strand with a short (left figures) or long 3’-mismatch tail (right figures), which must be removed before the remaining 3’ end can be extended to complement the HDRT insert region and its distal homology arm. We propose that the removal of this 3’ mismatch tail is a rate-limiting step determining editing efficiency when genomic sequences are replaced. ( B ) The predicted length of the 3’-mismatch tail in experiments presented in are plotted against the efficiency with which an HA-tag is introduced into the HCDR3 region, as determined by flow cytometry. Error bar indication SD from two independent experiments. ( C ) A comparison of editing efficiency between those with short (<10 nt) or long (>10 nt) 3’ mismatch tails. Editing by SpCas9 or Mb2Cas12a is significantly more efficient with short 3’ mismatch tails, as determined by one-way ANOVA with Tukey’s multiple comparison test (p<0.0001). Dots represent pool data from 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 plasmid numbers 69982, 69988, 69983, and 42230, respectively). pMAL-his-LbCpf1-EC was a gift from Dr. Jin-Soo Kim (Addgene plasmid number 79008) and was used to express Mb2Cas12a in E. coli for protein production.

Techniques: Genomic Sequencing, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: Efficient reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire

doi: 10.1101/2021.04.01.437943

Figure Lengend Snippet: ( 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. In addition the amino-acid translations of four HDRT used in the subsequent panels are represented in green, 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 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 panel B. flow cytometric studies used to generate panel 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 panel D were analyzed two 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). Grey 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 next-generation sequencing (NGS) of the VDJ region. Sequences were divided into four categories, depending on whether the edited sequence matched exactly the HDRT (Perfect HDR), whether 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 plasmid numbers 69982, 69988, 69983, and 42230, respectively). pMAL-his-LbCpf1-EC was a gift from Dr. Jin-Soo Kim (Addgene plasmid number 79008) and was used to express Mb2Cas12a in E. coli for protein production.

Techniques: Sequencing, Flow Cytometry, Labeling, Expressing, Derivative Assay, Mutagenesis, Control, Next-Generation Sequencing, Modification

Journal: bioRxiv

Article Title: Efficient reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire

doi: 10.1101/2021.04.01.437943

Figure Lengend Snippet: ( 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 edited 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 panel A, using primary B cells from three independent donors. Note that a mixture of three HDRT 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 indicted 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, quantified as described in except that the VH-family of edited cells 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 plasmid numbers 69982, 69988, 69983, and 42230, respectively). pMAL-his-LbCpf1-EC was a gift from Dr. Jin-Soo Kim (Addgene plasmid number 79008) and was used to express Mb2Cas12a in E. coli for protein production.

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

Journal: bioRxiv

Article Title: Efficient reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire

doi: 10.1101/2021.04.01.437943

Figure Lengend Snippet: Primary cells were electroporated with Mb2Cas12a RNP and HDRT encoding an HA tag ( A ) or the HCDR3 regions of the HIV-1 neutralizing antibodies CH01 ( B ) and PG9 ( C ), with the same mixture of homology arms as those used in . Cells were sorted with an anti-HA antibody (HA tag, panel A) or a SOSIP trimer derived from the CRF_AG_250 isolate (panels B and C). Edited cells were analyzed by NGS before and after sorting, and the frequency of each VH1-,VH3-, and VH4-family gene was measured. Flow cytometry histograms displays one of two experiments with similar results, and bar graphs indicate the mean of those two experiments. ( D ) Antibodies composed the heavy-chains expressed from the indicated VH genes enriched in panel C or that of PG9, the PG9 HCDR3, a transmembrane domain, and the native PG9 light chain were expressed on the surface of 293T cells and analyzed by flow cytometry. One of two representative experiments is presented. Mature indicates expression of the original PG9 antibody. ( E ) The mean of two experiments shown in panel D is presented. ( F ) The IC 50 values of soluble forms of the antibodies characterized in panel D against indicated HIV-1 isolates is represented.

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 plasmid numbers 69982, 69988, 69983, and 42230, respectively). pMAL-his-LbCpf1-EC was a gift from Dr. Jin-Soo Kim (Addgene plasmid number 79008) and was used to express Mb2Cas12a in E. coli for protein production.

Techniques: Derivative Assay, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Efficient reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire

doi: 10.1101/2021.04.01.437943

Figure Lengend Snippet: ( 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 3’ of a recombined V gene, a D gene, and the 5’ of a J-chain. To insert a common HCDR3 into a diverse population of BCR, the guide RNA (gRNA) of a CRISPR effector protein must complement a conserved HC region at the 3’ 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 3’ of the HCDR3-encoding sequence but oriented Cas12a cleavage within the 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 transfected with plasmids encoding BsCas12a, TsCas12a, Mb2Cas12a, or Mb3Cas12a. 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 indicted range of two independent experiments, and asterisks indicate statistical significance relative to controls. Statistical difference were determined by non-paired Students t-test, (****, p<0.0001). ( C ) Mb2Cas12 RNP were 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 inexact NHEJ. ( D ) Results of three experiments similar to that shown in panel C. Error bars indicate standard error (SEM). Asterisks indicted significant differences from the canonical TTTG PAM (Mb2Cas12a-RNP or AsCas12a, respectively). Statistical difference were determined by non-paired Students 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 plasmid numbers 69982, 69988, 69983, and 42230, respectively). pMAL-his-LbCpf1-EC was a gift from Dr. Jin-Soo Kim (Addgene plasmid number 79008) and was used to express Mb2Cas12a in E. coli for protein production.

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

Journal: bioRxiv

Article Title: Efficient reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire

doi: 10.1101/2021.04.01.437943

Figure Lengend Snippet: ( A ) A diagram representing four HDR templates (HDRT) used in panels B-E. Specifically, sense and anti-sense forms of HDRT-A, were used to replace a 9-nucleotide (nt) region (grey) with 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 antisense forms are represented in red and blue, respectively. SpCas9 (cyan) and Mb2Cas12 (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 five nucleotides. ( B ) A representative example of an experiment used to generate panels C-E in which editing efficiency of MbCas12A or SpCas9 RNP is 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 panel B, for all four sites shown in panel A. Differences between Mb2 and Cas9, and among the four sites, are not significant (n.s.). The same data generated for panel C was replotted according to whether the sense or anti-sense HDRT were 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 plasmid numbers 69982, 69988, 69983, and 42230, respectively). pMAL-his-LbCpf1-EC was a gift from Dr. Jin-Soo Kim (Addgene plasmid number 79008) and was used to express Mb2Cas12a in E. coli for protein production.

Techniques: Flow Cytometry, Knock-In, Generated

Journal: bioRxiv

Article Title: Efficient reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire

doi: 10.1101/2021.04.01.437943

Figure Lengend Snippet: ( A ) A model showing where a 3’ mismatch tail occurs. A cut site (yellow) is introduced into a region of the gene targeted for replacement (grey), asymmetrically dividing this region. Efficient 5’ to 3’ resection exposes two 3’ ends. An HDRT can complement a strand with a short (left figures) or long 3’-mismatch tail (right figures), which must be removed before the remaining 3’ end can be extended to complement the HDRT insert region and its distal homology arm. We propose that the removal of this 3’ mismatch tail is a rate-limiting step determining editing efficiency when genomic sequences are replaced. ( B ) The predicted length of the 3’-mismatch tail in experiments presented in are plotted against the efficiency with which an HA-tag is introduced into the HCDR3 region, as determined by flow cytometry. Error bar indication SD from two independent experiments. ( C ) A comparison of editing efficiency between those with short (<10 nt) or long (>10 nt) 3’ mismatch tails. Editing by SpCas9 or Mb2Cas12a is significantly more efficient with short 3’ mismatch tails, as determined by one-way ANOVA with Tukey’s multiple comparison test (p<0.0001). Dots represent pool data from 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 plasmid numbers 69982, 69988, 69983, and 42230, respectively). pMAL-his-LbCpf1-EC was a gift from Dr. Jin-Soo Kim (Addgene plasmid number 79008) and was used to express Mb2Cas12a in E. coli for protein production.

Techniques: Genomic Sequencing, Flow Cytometry

Journal: bioRxiv

Article Title: Efficient reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire

doi: 10.1101/2021.04.01.437943

Figure Lengend Snippet: ( 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. In addition the amino-acid translations of four HDRT used in the subsequent panels are represented in green, 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 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 panel B. flow cytometric studies used to generate panel 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 panel D were analyzed two 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). Grey 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 next-generation sequencing (NGS) of the VDJ region. Sequences were divided into four categories, depending on whether the edited sequence matched exactly the HDRT (Perfect HDR), whether 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 plasmid numbers 69982, 69988, 69983, and 42230, respectively). pMAL-his-LbCpf1-EC was a gift from Dr. Jin-Soo Kim (Addgene plasmid number 79008) and was used to express Mb2Cas12a in E. coli for protein production.

Techniques: Sequencing, Flow Cytometry, Labeling, Expressing, Derivative Assay, Mutagenesis, Next-Generation Sequencing, Modification

Journal: bioRxiv

Article Title: Efficient reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire

doi: 10.1101/2021.04.01.437943

Figure Lengend Snippet: ( 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 edited 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 panel A, using primary B cells from three independent donors. Note that a mixture of three HDRT 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 indicted 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, quantified as described in except that the VH-family of edited cells 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 plasmid numbers 69982, 69988, 69983, and 42230, respectively). pMAL-his-LbCpf1-EC was a gift from Dr. Jin-Soo Kim (Addgene plasmid number 79008) and was used to express Mb2Cas12a in E. coli for protein production.

Techniques: Flow Cytometry, Modification, Binding Assay, Sequencing

Journal: bioRxiv

Article Title: Efficient reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire

doi: 10.1101/2021.04.01.437943

Figure Lengend Snippet: Primary cells were electroporated with Mb2Cas12a RNP and HDRT encoding an HA tag ( A ) or the HCDR3 regions of the HIV-1 neutralizing antibodies CH01 ( B ) and PG9 ( C ), with the same mixture of homology arms as those used in . Cells were sorted with an anti-HA antibody (HA tag, panel A) or a SOSIP trimer derived from the CRF_AG_250 isolate (panels B and C). Edited cells were analyzed by NGS before and after sorting, and the frequency of each VH1-,VH3-, and VH4-family gene was measured. Flow cytometry histograms displays one of two experiments with similar results, and bar graphs indicate the mean of those two experiments. ( D ) Antibodies composed the heavy-chains expressed from the indicated VH genes enriched in panel C or that of PG9, the PG9 HCDR3, a transmembrane domain, and the native PG9 light chain were expressed on the surface of 293T cells and analyzed by flow cytometry. One of two representative experiments is presented. Mature indicates expression of the original PG9 antibody. ( E ) The mean of two experiments shown in panel D is presented. ( F ) The IC 50 values of soluble forms of the antibodies characterized in panel D against indicated HIV-1 isolates is represented.

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 plasmid numbers 69982, 69988, 69983, and 42230, respectively). pMAL-his-LbCpf1-EC was a gift from Dr. Jin-Soo Kim (Addgene plasmid number 79008) and was used to express Mb2Cas12a in E. coli for protein production.

Techniques: Derivative Assay, Flow Cytometry, Expressing