bsai hf  (New England Biolabs)


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    Structured Review

    New England Biolabs bsai hf
    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 <t>LacZ</t> cassette, derived from pUC19, flanked by <t>BsaI</t> 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 .
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    Images

    1) 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

    2) Product Images from "COSPLAY: An expandable toolbox for combinatorial and swift generation of expression plasmids in yeast"

    Article Title: COSPLAY: An expandable toolbox for combinatorial and swift generation of expression plasmids in yeast

    Journal: bioRxiv

    doi: 10.1101/630277

    Plasmid architecture in the COSPLAY toolbox A) Schematic representation of an expression vector assembled by COSPLAY composed of 6 independent modules. Each module represents a different type of sequence as indicated on the legend. B) Restriction site of the BsaI endonuclease and specific overhangs used; C) Principle of COSPLAY expression vector assembly. A destination vector (pDV) is mixed with 6 plasmids containing compatible module in a single tube. Expression vector is assembled through simultaneous digestion/ligation in the presence of BsaI and T4 DNA ligase. Specific overhangs that ensure that individual modules are assembled in the right order and orientation are indicated in pink.
    Figure Legend Snippet: Plasmid architecture in the COSPLAY toolbox A) Schematic representation of an expression vector assembled by COSPLAY composed of 6 independent modules. Each module represents a different type of sequence as indicated on the legend. B) Restriction site of the BsaI endonuclease and specific overhangs used; C) Principle of COSPLAY expression vector assembly. A destination vector (pDV) is mixed with 6 plasmids containing compatible module in a single tube. Expression vector is assembled through simultaneous digestion/ligation in the presence of BsaI and T4 DNA ligase. Specific overhangs that ensure that individual modules are assembled in the right order and orientation are indicated in pink.

    Techniques Used: Plasmid Preparation, Expressing, Sequencing, Ligation

    3) Product Images from "Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining"

    Article Title: Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

    Journal: bioRxiv

    doi: 10.1101/754242

    Golden Transformation method for ADP1 genome engineering. Two PCR reactions are performed to create upstream (U) and downstream (D) genomic target flanks with added terminal BsaI and BsmBI type IIS restriction sites as depicted. The two PCR products can then be combined via BsaI Golden Gate assembly (GGA) with the selection cassette to form a replacement DNA or combined with one another and optionally with additional genetic parts (not shown) via BsmBI GGA to form a rescue cassette. The positive-negative selection cassette ( tdk - kanR ) is maintained on the high-copy pBTK622 plasmid that has an origin that does not replicate in A. baylyi . The first GGA reaction is added to an A. baylyi culture and then plated on LB-Kan to select for transformants with the replacement cassette integrated into the genome. Then, transformation of the second assembly reaction with counterselection on LB-AZT is used to move the unmarked deletions/additions encoded on the rescue cassette into the genome.
    Figure Legend Snippet: Golden Transformation method for ADP1 genome engineering. Two PCR reactions are performed to create upstream (U) and downstream (D) genomic target flanks with added terminal BsaI and BsmBI type IIS restriction sites as depicted. The two PCR products can then be combined via BsaI Golden Gate assembly (GGA) with the selection cassette to form a replacement DNA or combined with one another and optionally with additional genetic parts (not shown) via BsmBI GGA to form a rescue cassette. The positive-negative selection cassette ( tdk - kanR ) is maintained on the high-copy pBTK622 plasmid that has an origin that does not replicate in A. baylyi . The first GGA reaction is added to an A. baylyi culture and then plated on LB-Kan to select for transformants with the replacement cassette integrated into the genome. Then, transformation of the second assembly reaction with counterselection on LB-AZT is used to move the unmarked deletions/additions encoded on the rescue cassette into the genome.

    Techniques Used: Transformation Assay, Polymerase Chain Reaction, Selection, Plasmid Preparation

    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 "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

    6) 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

    7) Product Images from "Pervasive off-target and double-stranded DNA nicking by CRISPR-Cas12a"

    Article Title: Pervasive off-target and double-stranded DNA nicking by CRISPR-Cas12a

    Journal: bioRxiv

    doi: 10.1101/657791

    Cas12a has activated non-specific, trans dsDNA nicking and degradation activity. (A) Representative agarose gel showing non-specific nicking and linearization of negatively supercoiled (nSC) dsDNA plasmid, and degradation of linearized and nicked dsDNA plasmid over time by LbCas12a. LbCas12a (20 nM) and crRNA (30 nM) were complexed with a crRNA-complementary 50 bp dsDNA activator (30 nM) (perfect target from pLibrary PS4) and incubated with non-specific dsDNA in different forms – negatively supercoiled (nSC), linear (li) and nicked (n). (B) Representative agarose gel showing target-dependent cleavage and degradation of dsDNA by LbCas12a. (C) Representative agarose gel showing RuvC-domain dependent pTarget linearization and activated non-specific nicking and degradation of dsDNA plasmid. Pre-crRNA processing and RuvC-domain active site mutants of LbCas12a (20 nM) and crRNA (30 nM) were complexed with a crRNA-complementary 50bp dsDNA activator (30 nM) (perfect target from pLibrary PS4) and incubated with non-specific dsDNA in different forms – negatively supercoiled (nSC), linear (li) and nicked (n). Time points at which the samples were collected are 5 min, 15 min, 30 min, 1 hour, 4 hours, 8 hours, and 24 hours. Controls: -cr = reaction without cognate crRNA, n = Nt.BspQI nicked pUC19, li = BsaI-HF linearized pUC19.
    Figure Legend Snippet: Cas12a has activated non-specific, trans dsDNA nicking and degradation activity. (A) Representative agarose gel showing non-specific nicking and linearization of negatively supercoiled (nSC) dsDNA plasmid, and degradation of linearized and nicked dsDNA plasmid over time by LbCas12a. LbCas12a (20 nM) and crRNA (30 nM) were complexed with a crRNA-complementary 50 bp dsDNA activator (30 nM) (perfect target from pLibrary PS4) and incubated with non-specific dsDNA in different forms – negatively supercoiled (nSC), linear (li) and nicked (n). (B) Representative agarose gel showing target-dependent cleavage and degradation of dsDNA by LbCas12a. (C) Representative agarose gel showing RuvC-domain dependent pTarget linearization and activated non-specific nicking and degradation of dsDNA plasmid. Pre-crRNA processing and RuvC-domain active site mutants of LbCas12a (20 nM) and crRNA (30 nM) were complexed with a crRNA-complementary 50bp dsDNA activator (30 nM) (perfect target from pLibrary PS4) and incubated with non-specific dsDNA in different forms – negatively supercoiled (nSC), linear (li) and nicked (n). Time points at which the samples were collected are 5 min, 15 min, 30 min, 1 hour, 4 hours, 8 hours, and 24 hours. Controls: -cr = reaction without cognate crRNA, n = Nt.BspQI nicked pUC19, li = BsaI-HF linearized pUC19.

    Techniques Used: Activity Assay, Agarose Gel Electrophoresis, Plasmid Preparation, Incubation

    High-throughput in vitro cleavage assay reveals target-dependent cleavage and nicking activity of Cas12a. (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 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 are 1 min, 5 min, 30 min, 1 hour, and 3 hours. Controls: -cr = reaction without cognate crRNA, n = Nt.BspQI nicked pUC19, li = BsaI-HF linearized pUC19 (C) Mismatch distribution of the pLibrary PS4 in the supercoiled and nicked fractions for different Cas12a orthologs. Depletion of target sequences from the supercoiled fraction indicates cleavage, and enrichment in the nicked fraction indicates nicking. The decrease in nicked fraction over time indicates linearization of target sequences. Error bars are SD, n = 3 replicates
    Figure Legend Snippet: High-throughput in vitro cleavage assay reveals target-dependent cleavage and nicking activity of Cas12a. (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 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 are 1 min, 5 min, 30 min, 1 hour, and 3 hours. Controls: -cr = reaction without cognate crRNA, n = Nt.BspQI nicked pUC19, li = BsaI-HF linearized pUC19 (C) Mismatch distribution of the pLibrary PS4 in the supercoiled and nicked fractions for different Cas12a orthologs. Depletion of target sequences from the supercoiled fraction indicates cleavage, and enrichment in the nicked fraction indicates nicking. The decrease in nicked fraction over time indicates linearization of target sequences. Error bars are SD, n = 3 replicates

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

    Cas12a orthologs have distinct nicking patterns against mismatched targets. (A) Representative agarose gels showing cleavage of a negatively supercoiled (nSC) plasmid containing the perfect target (pTarget) or mismatched (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 are 15 sec, 30 sec, 1 min, 2 min, 5 min, 15 min, 30 min, 1 hour, 3 hours, and 5 hours. Controls: -cr = reaction without cognate crRNA, n = Nt.BspQI nicked pUC19, li = BsaI-HF linearized pUC19. (B) Quantification of supercoiled, linear and nicked fractions from cleavage of perfect and mismatched (MM) target plasmid by LbCas12a after 3 hours. -/- indicates a cleavage reaction with the target plasmid without Cas12a and crRNA. -/+ indicates a cleavage reaction with the target plasmid and Cas12a only, and +/+ indicates a cleavage reaction with the target plasmid, Cas12a and cognate crRNA. Average of the intensity fraction values are plotted with SD as error bars, n = 3 replicates.
    Figure Legend Snippet: Cas12a orthologs have distinct nicking patterns against mismatched targets. (A) Representative agarose gels showing cleavage of a negatively supercoiled (nSC) plasmid containing the perfect target (pTarget) or mismatched (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 are 15 sec, 30 sec, 1 min, 2 min, 5 min, 15 min, 30 min, 1 hour, 3 hours, and 5 hours. Controls: -cr = reaction without cognate crRNA, n = Nt.BspQI nicked pUC19, li = BsaI-HF linearized pUC19. (B) Quantification of supercoiled, linear and nicked fractions from cleavage of perfect and mismatched (MM) target plasmid by LbCas12a after 3 hours. -/- indicates a cleavage reaction with the target plasmid without Cas12a and crRNA. -/+ indicates a cleavage reaction with the target plasmid and Cas12a only, and +/+ indicates a cleavage reaction with the target plasmid, Cas12a and cognate crRNA. Average of the intensity fraction values are plotted with SD as error bars, n = 3 replicates.

    Techniques Used: Plasmid Preparation

    8) 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

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    New England Biolabs bsai hf
    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 <t>LacZ</t> cassette, derived from pUC19, flanked by <t>BsaI</t> 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 .
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    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 .

    Journal: bioRxiv

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

    doi: 10.1101/2021.04.20.440701

    Figure Lengend 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 .

    Article Snippet: The amplified backbone was digested with BsaI-HF (NEB) and the LacZ insert was digested with BbsI-HF (NEB).

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

    Plasmid architecture in the COSPLAY toolbox A) Schematic representation of an expression vector assembled by COSPLAY composed of 6 independent modules. Each module represents a different type of sequence as indicated on the legend. B) Restriction site of the BsaI endonuclease and specific overhangs used; C) Principle of COSPLAY expression vector assembly. A destination vector (pDV) is mixed with 6 plasmids containing compatible module in a single tube. Expression vector is assembled through simultaneous digestion/ligation in the presence of BsaI and T4 DNA ligase. Specific overhangs that ensure that individual modules are assembled in the right order and orientation are indicated in pink.

    Journal: bioRxiv

    Article Title: COSPLAY: An expandable toolbox for combinatorial and swift generation of expression plasmids in yeast

    doi: 10.1101/630277

    Figure Lengend Snippet: Plasmid architecture in the COSPLAY toolbox A) Schematic representation of an expression vector assembled by COSPLAY composed of 6 independent modules. Each module represents a different type of sequence as indicated on the legend. B) Restriction site of the BsaI endonuclease and specific overhangs used; C) Principle of COSPLAY expression vector assembly. A destination vector (pDV) is mixed with 6 plasmids containing compatible module in a single tube. Expression vector is assembled through simultaneous digestion/ligation in the presence of BsaI and T4 DNA ligase. Specific overhangs that ensure that individual modules are assembled in the right order and orientation are indicated in pink.

    Article Snippet: Reaction conditions: 1X NEB® Buffer 4, 1mM ATP, 20U BsaI-HF (NEB®), 400U T4 DNA ligase (NEB®) and H20 qsp 20μl.

    Techniques: Plasmid Preparation, Expressing, Sequencing, Ligation

    Golden Transformation method for ADP1 genome engineering. Two PCR reactions are performed to create upstream (U) and downstream (D) genomic target flanks with added terminal BsaI and BsmBI type IIS restriction sites as depicted. The two PCR products can then be combined via BsaI Golden Gate assembly (GGA) with the selection cassette to form a replacement DNA or combined with one another and optionally with additional genetic parts (not shown) via BsmBI GGA to form a rescue cassette. The positive-negative selection cassette ( tdk - kanR ) is maintained on the high-copy pBTK622 plasmid that has an origin that does not replicate in A. baylyi . The first GGA reaction is added to an A. baylyi culture and then plated on LB-Kan to select for transformants with the replacement cassette integrated into the genome. Then, transformation of the second assembly reaction with counterselection on LB-AZT is used to move the unmarked deletions/additions encoded on the rescue cassette into the genome.

    Journal: bioRxiv

    Article Title: Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

    doi: 10.1101/754242

    Figure Lengend Snippet: Golden Transformation method for ADP1 genome engineering. Two PCR reactions are performed to create upstream (U) and downstream (D) genomic target flanks with added terminal BsaI and BsmBI type IIS restriction sites as depicted. The two PCR products can then be combined via BsaI Golden Gate assembly (GGA) with the selection cassette to form a replacement DNA or combined with one another and optionally with additional genetic parts (not shown) via BsmBI GGA to form a rescue cassette. The positive-negative selection cassette ( tdk - kanR ) is maintained on the high-copy pBTK622 plasmid that has an origin that does not replicate in A. baylyi . The first GGA reaction is added to an A. baylyi culture and then plated on LB-Kan to select for transformants with the replacement cassette integrated into the genome. Then, transformation of the second assembly reaction with counterselection on LB-AZT is used to move the unmarked deletions/additions encoded on the rescue cassette into the genome.

    Article Snippet: Golden Gate assembly DNA fragments for transformation were constructed using Golden Gate Assembly (GGA) reactions containing 1 U/µl BsaI-HF or 0.5 U/µl BsmBI and 150 U/µl of either T7 or T4 DNA ligase in T4 DNA ligase buffer (New England Biolabs) ( , ).

    Techniques: Transformation Assay, Polymerase Chain Reaction, Selection, Plasmid Preparation

    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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkab163

    Figure Lengend 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

    Article Snippet: For controls, pUC19 was prepared by restriction enzyme digestion using BsaI-HF to linearize the plasmid and Nt.BspQI to nick the plasmid using the manufacturer's protocols (New England Biolabs).

    Techniques: Plasmid Preparation, Incubation