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    Structured Review

    New England Biolabs bsai hf
    Design of high-throughput pathway refactoring. a Refactoring scheme for a representative RiPP BGC using Golden Gate assembly. Genes involved in transcriptional regulation, leader peptide removal, and cellular export were omitted from refactoring. The codon-optimized gene encoding the precursor peptide was inserted into helper plasmid 1 with an N-terminal His 6 -tag and other genes encoding biosynthetic enzymes were inserted into the following helper plasmids. Each helper plasmid contained two Bsa I recognition sites flanking the insert, followed by a unique pair of 4-bp linkers, a T7 promoter, and a T7 terminator. The constructed cassettes that comprise of a gene driven by a T7 promoter in the helper plasmid can then be assembled in a defined order into the lacZ -containing receiver plasmid with pET28a backbone in a single step via <t>BsaI</t> -catalyzed Golden Gate assembly. The transformants were assessed by blue-white screening and further confirmed by <t>Sanger</t> <t>DNA</t> sequencing. b Workflow for high-throughput refactoring of 96 RiPP BGCs. Labcyte Echo 550 was used for setting up 96 Golden Gate reactions and Tecan FluentControl 1080 was used for E. coli transformation and plasmid extraction. N represents the number of constructs evaluated for each refactored RiPP BGC. Figure 2b was created with BioRender.
    Bsai Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bsai hf - by Bioz Stars, 2022-12
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    Images

    1) Product Images from "A scalable platform to discover antimicrobials of ribosomal origin"

    Article Title: A scalable platform to discover antimicrobials of ribosomal origin

    Journal: Nature Communications

    doi: 10.1038/s41467-022-33890-w

    Design of high-throughput pathway refactoring. a Refactoring scheme for a representative RiPP BGC using Golden Gate assembly. Genes involved in transcriptional regulation, leader peptide removal, and cellular export were omitted from refactoring. The codon-optimized gene encoding the precursor peptide was inserted into helper plasmid 1 with an N-terminal His 6 -tag and other genes encoding biosynthetic enzymes were inserted into the following helper plasmids. Each helper plasmid contained two Bsa I recognition sites flanking the insert, followed by a unique pair of 4-bp linkers, a T7 promoter, and a T7 terminator. The constructed cassettes that comprise of a gene driven by a T7 promoter in the helper plasmid can then be assembled in a defined order into the lacZ -containing receiver plasmid with pET28a backbone in a single step via BsaI -catalyzed Golden Gate assembly. The transformants were assessed by blue-white screening and further confirmed by Sanger DNA sequencing. b Workflow for high-throughput refactoring of 96 RiPP BGCs. Labcyte Echo 550 was used for setting up 96 Golden Gate reactions and Tecan FluentControl 1080 was used for E. coli transformation and plasmid extraction. N represents the number of constructs evaluated for each refactored RiPP BGC. Figure 2b was created with BioRender.
    Figure Legend Snippet: Design of high-throughput pathway refactoring. a Refactoring scheme for a representative RiPP BGC using Golden Gate assembly. Genes involved in transcriptional regulation, leader peptide removal, and cellular export were omitted from refactoring. The codon-optimized gene encoding the precursor peptide was inserted into helper plasmid 1 with an N-terminal His 6 -tag and other genes encoding biosynthetic enzymes were inserted into the following helper plasmids. Each helper plasmid contained two Bsa I recognition sites flanking the insert, followed by a unique pair of 4-bp linkers, a T7 promoter, and a T7 terminator. The constructed cassettes that comprise of a gene driven by a T7 promoter in the helper plasmid can then be assembled in a defined order into the lacZ -containing receiver plasmid with pET28a backbone in a single step via BsaI -catalyzed Golden Gate assembly. The transformants were assessed by blue-white screening and further confirmed by Sanger DNA sequencing. b Workflow for high-throughput refactoring of 96 RiPP BGCs. Labcyte Echo 550 was used for setting up 96 Golden Gate reactions and Tecan FluentControl 1080 was used for E. coli transformation and plasmid extraction. N represents the number of constructs evaluated for each refactored RiPP BGC. Figure 2b was created with BioRender.

    Techniques Used: High Throughput Screening Assay, Plasmid Preparation, Construct, DNA Sequencing, Transformation Assay

    2) Product Images from "Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection"

    Article Title: Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection

    Journal: bioRxiv

    doi: 10.1101/2021.04.20.440701

    Cloning methodology. (A) A diagram showing the steps of generating pNHT7, the base vector used to clone endogenous planarian genes. The overall design is a LacZ cassette, derived from pUC19, flanked by BsaI restriction sites, which allow for subcloning of planarian genes into pNHT7. Upstream is a T7 promoter, which allows for in vitro transcription (IVT), and all of these are flanked by M13 forward and reverse primers which are used to generate linearized templates for IVT through PCR. The backbone for pNHT7 is derived from the backbone from pDONOR221. (B) Isolating the 5’ and 3’ UTRs of a gene of interest (GOI) from cDNA uses primers containing pNHT7 compatible BsaI restriction sites followed by 5’ and 3’ UTR sequences of the GOI. After amplification, the fragment containing the GOI can be cloned into pNHT7 though a standard golden gate reaction. (C) The Nluc reporter is inserted between the UTR sequences by using primers containing BsaI restriction sites to prime the 5’ and 3’ ends of the reporter as well as outward facing primers which add complementary BsaI restriction sites and prime the end of the 5’ UTR and beginning of the 3’ UTR for the gene captured in pNHT7. These two amplicons can then be purified and assembled via a golden gate reaction. All primer sequences are provided in Supplementary Table 4 .
    Figure Legend Snippet: Cloning methodology. (A) A diagram showing the steps of generating pNHT7, the base vector used to clone endogenous planarian genes. The overall design is a LacZ cassette, derived from pUC19, flanked by BsaI restriction sites, which allow for subcloning of planarian genes into pNHT7. Upstream is a T7 promoter, which allows for in vitro transcription (IVT), and all of these are flanked by M13 forward and reverse primers which are used to generate linearized templates for IVT through PCR. The backbone for pNHT7 is derived from the backbone from pDONOR221. (B) Isolating the 5’ and 3’ UTRs of a gene of interest (GOI) from cDNA uses primers containing pNHT7 compatible BsaI restriction sites followed by 5’ and 3’ UTR sequences of the GOI. After amplification, the fragment containing the GOI can be cloned into pNHT7 though a standard golden gate reaction. (C) The Nluc reporter is inserted between the UTR sequences by using primers containing BsaI restriction sites to prime the 5’ and 3’ ends of the reporter as well as outward facing primers which add complementary BsaI restriction sites and prime the end of the 5’ UTR and beginning of the 3’ UTR for the gene captured in pNHT7. These two amplicons can then be purified and assembled via a golden gate reaction. All primer sequences are provided in Supplementary Table 4 .

    Techniques Used: Clone Assay, Plasmid Preparation, Derivative Assay, Subcloning, In Vitro, Polymerase Chain Reaction, Amplification, Purification

    3) Product Images from "Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection"

    Article Title: Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection

    Journal: bioRxiv

    doi: 10.1101/2021.04.20.440701

    Cloning methodology. (A) A diagram showing the steps of generating pNHT7, the base vector used to clone endogenous planarian genes. The overall design is a LacZ cassette, derived from pUC19, flanked by BsaI restriction sites, which allow for subcloning of planarian genes into pNHT7. Upstream is a T7 promoter, which allows for in vitro transcription (IVT), and all of these are flanked by M13 forward and reverse primers which are used to generate linearized templates for IVT through PCR. The backbone for pNHT7 is derived from the backbone from pDONOR221. (B) Isolating the 5’ and 3’ UTRs of a gene of interest (GOI) from cDNA uses primers containing pNHT7 compatible BsaI restriction sites followed by 5’ and 3’ UTR sequences of the GOI. After amplification, the fragment containing the GOI can be cloned into pNHT7 though a standard golden gate reaction. (C) The Nluc reporter is inserted between the UTR sequences by using primers containing BsaI restriction sites to prime the 5’ and 3’ ends of the reporter as well as outward facing primers which add complementary BsaI restriction sites and prime the end of the 5’ UTR and beginning of the 3’ UTR for the gene captured in pNHT7. These two amplicons can then be purified and assembled via a golden gate reaction. All primer sequences are provided in Supplementary Table 4 .
    Figure Legend Snippet: Cloning methodology. (A) A diagram showing the steps of generating pNHT7, the base vector used to clone endogenous planarian genes. The overall design is a LacZ cassette, derived from pUC19, flanked by BsaI restriction sites, which allow for subcloning of planarian genes into pNHT7. Upstream is a T7 promoter, which allows for in vitro transcription (IVT), and all of these are flanked by M13 forward and reverse primers which are used to generate linearized templates for IVT through PCR. The backbone for pNHT7 is derived from the backbone from pDONOR221. (B) Isolating the 5’ and 3’ UTRs of a gene of interest (GOI) from cDNA uses primers containing pNHT7 compatible BsaI restriction sites followed by 5’ and 3’ UTR sequences of the GOI. After amplification, the fragment containing the GOI can be cloned into pNHT7 though a standard golden gate reaction. (C) The Nluc reporter is inserted between the UTR sequences by using primers containing BsaI restriction sites to prime the 5’ and 3’ ends of the reporter as well as outward facing primers which add complementary BsaI restriction sites and prime the end of the 5’ UTR and beginning of the 3’ UTR for the gene captured in pNHT7. These two amplicons can then be purified and assembled via a golden gate reaction. All primer sequences are provided in Supplementary Table 4 .

    Techniques Used: Clone Assay, Plasmid Preparation, Derivative Assay, Subcloning, In Vitro, Polymerase Chain Reaction, Amplification, Purification

    4) Product Images from "Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR–Cas9 variants"

    Article Title: Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR–Cas9 variants

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab163

    Cas9 variants have different cleavage activities against mismatched targets. ( A ) Representative agarose gels showing cleavage of a negatively supercoiled (nSC) plasmid containing the perfect target (0 MM) or mismatched (2 to 5 MM) target over a time course by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 15 s, 30 s, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h and 5 h. tr:crRNA = tracrRNA:crRNA. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (–) = pTarget or pLibrary alone incubated at 37°C for the longest time point in the assay (5 h); (-cr) = pTarget or pLibrary incubated with Cas9 only at 37°C for the longest time point in the assay (5 h); n = Nt.BspQI nicked pUC19; li = BsaI-HF linearized pUC19. ( B ) Quantification of supercoiled, linear and nicked pools from cleavage of perfect or fully crRNA-complementary (0 MM) and mismatched (2 to 5 MM) target plasmid by Cas9 after 10 min and 3 h. pTarget MM indicates target plasmid (0, 2 to 5 MM) alone incubated at 37°C for the time points indicated. Target sequences tested are listed with PAM (bold) and mismatches (lowercase and red) indicated. (–) indicates a cleavage reaction with the target plasmid and Cas9 only, and (+) indicates a cleavage reaction with the target plasmid, Cas9 and cognate tracrRNA:crRNA. Values plotted represent an average of three replicates. Error bars are SEM. * or • indicate P
    Figure Legend Snippet: Cas9 variants have different cleavage activities against mismatched targets. ( A ) Representative agarose gels showing cleavage of a negatively supercoiled (nSC) plasmid containing the perfect target (0 MM) or mismatched (2 to 5 MM) target over a time course by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 15 s, 30 s, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h and 5 h. tr:crRNA = tracrRNA:crRNA. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (–) = pTarget or pLibrary alone incubated at 37°C for the longest time point in the assay (5 h); (-cr) = pTarget or pLibrary incubated with Cas9 only at 37°C for the longest time point in the assay (5 h); n = Nt.BspQI nicked pUC19; li = BsaI-HF linearized pUC19. ( B ) Quantification of supercoiled, linear and nicked pools from cleavage of perfect or fully crRNA-complementary (0 MM) and mismatched (2 to 5 MM) target plasmid by Cas9 after 10 min and 3 h. pTarget MM indicates target plasmid (0, 2 to 5 MM) alone incubated at 37°C for the time points indicated. Target sequences tested are listed with PAM (bold) and mismatches (lowercase and red) indicated. (–) indicates a cleavage reaction with the target plasmid and Cas9 only, and (+) indicates a cleavage reaction with the target plasmid, Cas9 and cognate tracrRNA:crRNA. Values plotted represent an average of three replicates. Error bars are SEM. * or • indicate P

    Techniques Used: Plasmid Preparation, Incubation

    5) Product Images from "High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants"

    Article Title: High-throughput in vitro specificity profiling of natural and high-fidelity CRISPR-Cas9 variants

    Journal: bioRxiv

    doi: 10.1101/2020.05.12.091991

    Cas9 variants have different cleavage activities against mismatched targets. (A) Representative agarose gels showing cleavage of a negatively supercoiled (nSC) plasmid containing the perfect target (0 MM) or mismatched (2 to 5 MM) target over a time course by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 15 sec, 30 sec, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h, and 5 h. tr:crRNA = tracrRNA:crRNA. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (−) = pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (5 h); (-cr) = pTarget or pLibrary incubated with Cas9 only at 37°C for the longest time point in the assay (5 h); n = Nt.BspQI nicked pUC19; li = BsaI-HF linearized pUC19 (B) Quantification of supercoiled, linear and nicked pools from cleavage of perfect or fully crRNA-complementary (0 MM) and mismatched (2 to 5 MM) target plasmid by Cas9 after 10 minutes and 3 hours. pTarget MM indicates target plasmid (0, 2 to 5 MM) alone incubated at 37 °C for the time points indicated. (−) indicates a cleavage reaction with the target plasmid and Cas9 only, and (+) indicates a cleavage reaction with the target plasmid, Cas9 and cognate tracrRNA:crRNA. Values plotted represent an average of three replicates. Error bars are SEM. The different target sequences tested are listed where the PAM is in bold and mismatches are in lowercase and red.
    Figure Legend Snippet: Cas9 variants have different cleavage activities against mismatched targets. (A) Representative agarose gels showing cleavage of a negatively supercoiled (nSC) plasmid containing the perfect target (0 MM) or mismatched (2 to 5 MM) target over a time course by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 15 sec, 30 sec, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h, and 5 h. tr:crRNA = tracrRNA:crRNA. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (−) = pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (5 h); (-cr) = pTarget or pLibrary incubated with Cas9 only at 37°C for the longest time point in the assay (5 h); n = Nt.BspQI nicked pUC19; li = BsaI-HF linearized pUC19 (B) Quantification of supercoiled, linear and nicked pools from cleavage of perfect or fully crRNA-complementary (0 MM) and mismatched (2 to 5 MM) target plasmid by Cas9 after 10 minutes and 3 hours. pTarget MM indicates target plasmid (0, 2 to 5 MM) alone incubated at 37 °C for the time points indicated. (−) indicates a cleavage reaction with the target plasmid and Cas9 only, and (+) indicates a cleavage reaction with the target plasmid, Cas9 and cognate tracrRNA:crRNA. Values plotted represent an average of three replicates. Error bars are SEM. The different target sequences tested are listed where the PAM is in bold and mismatches are in lowercase and red.

    Techniques Used: Plasmid Preparation, Incubation

    High-throughput in vitro analysis of Cas9 mismatch tolerance. (A) Outline and workflow of the high-throughput in vitro cleavage assay. (B) Representative agarose gel showing time course cleavage of negatively supercoiled (nSC) plasmid containing a fully matched PS4 target (pTarget PS4, left) and plasmid library PS4 (pLibrary PS4, right) by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 1 min, 5 min, 30 min, 1 h, and 3 h. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (−) = pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (3 h); (-r) = pTarget or pLibrary incubated with Cas9 only at 37 °C for the longest time point in the assay (3 h); n = Nt.BspQI nicked pUC19; li = BsaI-HF linearized pUC19 (C) Overall cleavage of the pLibrary PS4 by Cas9 indicating the decrease in supercoiled (nSC) pool and appearance of nicked (n) and linear (li) pools over time. The 0 time point is the quantification of the negative control pLibrary (i.e. pLibrary run on a gel after preparation as represented in Fig. S2A). Values plotted represent an average of two replicates. Error bars are SEM.
    Figure Legend Snippet: High-throughput in vitro analysis of Cas9 mismatch tolerance. (A) Outline and workflow of the high-throughput in vitro cleavage assay. (B) Representative agarose gel showing time course cleavage of negatively supercoiled (nSC) plasmid containing a fully matched PS4 target (pTarget PS4, left) and plasmid library PS4 (pLibrary PS4, right) by Cas9 variants, resulting in linear (li) and/or nicked (n) products. Time points at which the samples were collected are 1 min, 5 min, 30 min, 1 h, and 3 h. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls: (−) = pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (3 h); (-r) = pTarget or pLibrary incubated with Cas9 only at 37 °C for the longest time point in the assay (3 h); n = Nt.BspQI nicked pUC19; li = BsaI-HF linearized pUC19 (C) Overall cleavage of the pLibrary PS4 by Cas9 indicating the decrease in supercoiled (nSC) pool and appearance of nicked (n) and linear (li) pools over time. The 0 time point is the quantification of the negative control pLibrary (i.e. pLibrary run on a gel after preparation as represented in Fig. S2A). Values plotted represent an average of two replicates. Error bars are SEM.

    Techniques Used: High Throughput Screening Assay, In Vitro, Cleavage Assay, Agarose Gel Electrophoresis, Plasmid Preparation, Incubation, Negative Control

    6) Product Images from "CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects"

    Article Title: CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA120.012933

    High-throughput in vitro analysis of Cas12a mismatch tolerance. A , outline and workflow of the high-throughput in vitro cleavage assay. B , representative agarose gel showing time course cleavage of nSC plasmid containing a fully matched target (pTarget; left ) and plasmid library (pLibrary; right ) pLibrary PS4 by LbCas12a, resulting in linear ( li ) and/or nicked ( n ) products. Time points at which the samples were collected were 1 min, 5 min, 30 min, 1 h, and 3 h. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls were as follows: − cr , pTarget or pLibrary incubated with Cas12a only at 37 °C for the longest time point in the assay (3 h); (−), pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (3 h); n , Nt.BspQI nicked pUC19; li , BsaI-HF linearized pUC19. C , overall cleavage of the pLibrary by Cas12a, indicating the decrease in supercoiled ( nSC ) pool and appearance of nicked ( n ) and linear ( li ) pools over time. The zero time point is quantification of the (−) control shown in B . Error bars , S.D.; n = 2 for LbCas12a, n = 3 for FnCas12a and AsCas12a. D and E , normalized counts relative to the negative control (−) for targets sequences with different number of MM plotted against time in a log scale for pLibrary PS4 in the supercoiled ( D ) and nicked pool ( E ) for different Cas12a orthologs. Fn , FnCas12a; Lb , LbCas12a; As , AsCas12a. Error bars , propagation of S.E.; n = 2 for LbCas12a, n = 3 for FnCas12a and AsCas12a.
    Figure Legend Snippet: High-throughput in vitro analysis of Cas12a mismatch tolerance. A , outline and workflow of the high-throughput in vitro cleavage assay. B , representative agarose gel showing time course cleavage of nSC plasmid containing a fully matched target (pTarget; left ) and plasmid library (pLibrary; right ) pLibrary PS4 by LbCas12a, resulting in linear ( li ) and/or nicked ( n ) products. Time points at which the samples were collected were 1 min, 5 min, 30 min, 1 h, and 3 h. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls were as follows: − cr , pTarget or pLibrary incubated with Cas12a only at 37 °C for the longest time point in the assay (3 h); (−), pTarget or pLibrary alone incubated at 37 °C for the longest time point in the assay (3 h); n , Nt.BspQI nicked pUC19; li , BsaI-HF linearized pUC19. C , overall cleavage of the pLibrary by Cas12a, indicating the decrease in supercoiled ( nSC ) pool and appearance of nicked ( n ) and linear ( li ) pools over time. The zero time point is quantification of the (−) control shown in B . Error bars , S.D.; n = 2 for LbCas12a, n = 3 for FnCas12a and AsCas12a. D and E , normalized counts relative to the negative control (−) for targets sequences with different number of MM plotted against time in a log scale for pLibrary PS4 in the supercoiled ( D ) and nicked pool ( E ) for different Cas12a orthologs. Fn , FnCas12a; Lb , LbCas12a; As , AsCas12a. Error bars , propagation of S.E.; n = 2 for LbCas12a, n = 3 for FnCas12a and AsCas12a.

    Techniques Used: High Throughput Screening Assay, In Vitro, Cleavage Assay, Agarose Gel Electrophoresis, Plasmid Preparation, Incubation, Negative Control

    Cas12a orthologs have distinct nicking patterns against mismatched targets. A , representative agarose gels showing cleavage of nSC plasmid containing the perfect target ( pTarget ) or MM target over a time course by Cas12a orthologs, FnCas12a ( left ), LbCas12a ( center ), and AsCas12a ( right ), resulting in linear ( li ) and/or nicked ( n ) products. Time points at which the samples were collected were 15 s, 30 s, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h, and 5 h. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls were as follows: − cr , target plasmid incubated with Cas12a only at 37 °C for the longest time point in the assay (5 h); (−), target plasmid alone incubated at 37 °C for the longest time point in the assay (5 h); n , Nt.BspQI nicked pUC19; li , BsaI-HF linearized pUC19. B , quantification of supercoiled, linear, and nicked fractions from cleavage of perfect or fully crRNA-complementary and MM target plasmid by LbCas12a after 3 h. The different target sequences tested are listed where the PAM is in boldface and mismatches are in lowercase and red . −/−, a cleavage reaction with the target plasmid without Cas12a and crRNA; −/+, a cleavage reaction with the target plasmid and Cas12a only; +/+, a cleavage reaction with the target plasmid, Cas12a, and cognate crRNA. Averages of the intensity fraction values are plotted with S.D. ( error bars ); n = 3 replicates.
    Figure Legend Snippet: Cas12a orthologs have distinct nicking patterns against mismatched targets. A , representative agarose gels showing cleavage of nSC plasmid containing the perfect target ( pTarget ) or MM target over a time course by Cas12a orthologs, FnCas12a ( left ), LbCas12a ( center ), and AsCas12a ( right ), resulting in linear ( li ) and/or nicked ( n ) products. Time points at which the samples were collected were 15 s, 30 s, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h, 3 h, and 5 h. All controls were performed under the same conditions as the longest time point for the experimental samples. Controls were as follows: − cr , target plasmid incubated with Cas12a only at 37 °C for the longest time point in the assay (5 h); (−), target plasmid alone incubated at 37 °C for the longest time point in the assay (5 h); n , Nt.BspQI nicked pUC19; li , BsaI-HF linearized pUC19. B , quantification of supercoiled, linear, and nicked fractions from cleavage of perfect or fully crRNA-complementary and MM target plasmid by LbCas12a after 3 h. The different target sequences tested are listed where the PAM is in boldface and mismatches are in lowercase and red . −/−, a cleavage reaction with the target plasmid without Cas12a and crRNA; −/+, a cleavage reaction with the target plasmid and Cas12a only; +/+, a cleavage reaction with the target plasmid, Cas12a, and cognate crRNA. Averages of the intensity fraction values are plotted with S.D. ( error bars ); n = 3 replicates.

    Techniques Used: Plasmid Preparation, Incubation

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    New England Biolabs bsai hf v2
    Bsai Hf V2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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