py004  (Addgene inc)

Journal: Nature Communications

Article Title: Highly parallel profiling of the activities and specificities of Cas12a variants in human cells

doi: 10.1038/s41467-025-57150-9

Figure Lengend Snippet: a PAM compatibilities of the Cas12a variants in human cells. A heat map showing the average indel frequencies induced by the Cas12a variants at the 15 PAMs that are currently known to be recognized by Cas12a. b Pearson correlation coefficients between the Cas12a variant induced indel frequencies measured at the target sequences containing the same protospacers 7 days after transduction of Cas12a variant lentivirus into libraries integrated HEK293T cells. c Scatter plots showing the correlations of the five cases that are labeled as 1, 2, 3, 4 and 5 in the b . The Spearman correlation coefficient ( ρ ) and the Pearson correlation coefficient ( r ) are shown. In b and c , 15 PAM sequences were used for the analyses. d Comparison of the frequency of indels induced by the Cas12a variants at TTTA, TTTC, TTTG PAM; The boxes represent the 25th, 50th, and 75th percentiles, and the whiskers represent the 10th and 90th percentiles. The numbers of analyzed target sequences ( n ) are as follows: n = 1195, 1223, 1224, 1157, 1191, 1178, 1171 for TTTA, n = 1198, 1642, 1621, 1560, 1591, 1585, 1569 for TTTC, n = 1711, 1753, 1729, 1682, 1706, 1694, 1687 for TTTG (As variants); n = 1240, 1232 for TTTA, n = 1649, 1645, n = 1762, 1775 for TTTG (Eb variants); n = 1295, 1283 for TTTA, n = 1675, 1674, n = 1809, 1805 for TTTG (Lb2 variants); n = 1250 for TTTA, n = 1653 for TTTC, n = 1780 for TTTG (CeCas12a); n = 1202, 1216, 1244, 1300, 1243, 1199, 1210, 1165, 1174 for TTTA, n = 1629, 1623, 1682, 1615, 1646, 1647, 1597, 1596, 1623 for TTTC, n = 1753, 1755, 1809, 1738, 1780, 1778, 1699, 1697, 1748 for TTTG (LbCas12a variants); n = 1205, 1224, 1201 for TTTA, n = 1622, 1632, 1618 for TTTC, n = 1742, 1761, 1745 for TTTG (FnCas12a variants); paired t -test, two tailed. Source data are provided as a Source Data file.

Article Snippet: AsCas12a (Addgene, #69982), LbCas12a (Addgene, #69988), FnCas12a (Addgene, #69976), Lb2Cas12a (Addgene, #69983) human expression plasmids were purchased from the non-profit plasmid repository Addgene.

Techniques: Variant Assay, Transduction, Labeling, Comparison, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: DNA target binding-induced pre-crRNA processing in type II and V CRISPR-Cas systems

doi: 10.1093/nar/gkae1241

Figure Lengend Snippet: Target DNA binding activates cutting of pre-crRNA spacers. ( A ) Time-course assays of the target dsDNA (TS and NTS) cis -cleavage activity of FnCas12a guided by crRNA and pre-crRNA. Data are shown as mean ± SD ( n = 3 independent experiments). ( B ) Fluorescence time-course assays of the ssDNA reporter trans -cleavage activity of FnCas12a with crRNA and pre-crRNA. ( C ) Comparison of slopes of curves within 21–30 min from the time-course graph (panel A). The slope was calculated by simple linear regression. Data are shown as mean ± SD ( n = 3 independent experiments). Statistical analysis was done by two-tailed t -test. ( D ) Sequences of the pre-crRNA and target DNA used for Cas12a-catalyzed pre-crRNA cleavage assays. The pre-crRNAs are labeled with Cy5 at the 3′-end. The natural spacer lengths of crRNA in CRISPR-Cas12a systems are 26–32 nt. ( E ) Denaturing gel showing (left panel) FnCas12a-, (center panel) LbCas12a- and (right panel) AsCas12a-catalyzed cleavage of pre-crRNA spacers. ( F ) Sequences of the pre-crRNA1, tracrRNA and target DNA1 used for AacCas12b-catalyzed pre-crRNA cleavage assays. The pre-crRNA1 is labeled with Cy5 at the 3′-end. The natural spacer length of crRNA in CRISPR-AacCas12b system is 34–38 nt. ( G ) Denaturing gel showing AacCas12b-catalyzed cleavage of pre-crRNA1 spacer. ( H ) Sequences of the pre-crRNA, tracrRNA and target DNA used for SauCas9-catalyzed pre-crRNA cleavage assays. The pre-crRNA is labeled with Cy5 at the 5′-end. The natural spacer length of crRNA in CRISPR-SauCas9 system is 30 nt. ( I ) Denaturing gel showing SauCas9-catalyzed cleavage of pre-crRNA spacer. The arrows indicate cleavage sites. All assays were repeated three times independently with similar results (panels E, G and I).

Article Snippet: The protein-coding sequences of Francisella novicida U112 Cas12a (FnCas12a) (Addgene, Cat: 69976), Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) (Addgene, Cat: 69988), Acidaminococcus sp. Cas12a (AsCas12a) (Addgene, Cat: 69982), Cas12j (Addgene, Cat: 158801), Cas12i and Staphylococcus aureus Cas9 (SauCas9) (synthesized by SYNBIOB GENE) were separately subcloned into modified pET28a-His 6 -SUMO vector using a ClonExpress II One Step Cloning kit (Vazyme, Cat: C112-02).

Techniques: Binding Assay, Activity Assay, Fluorescence, Comparison, Two Tailed Test, Labeling, CRISPR

Journal: Nucleic Acids Research

Article Title: DNA target binding-induced pre-crRNA processing in type II and V CRISPR-Cas systems

doi: 10.1093/nar/gkae1241

Figure Lengend Snippet: Kinetic analysis of Cas12a-catalyzed pre-crRNA spacer cleavage. ( A ) Time-course assays of the pre-crRNA spacer cleavage activity of FnCas12a activated by target ssDNA, nontarget ssDNA, target dsDNA and target ssRNA activators. Data are shown as mean ± SD ( n = 3 independent experiments). ( B ) Effect of target ssDNA length on pre-crRNA cleavage activity of (left panel) FnCas12a, (center panel) LbCas12a and (right panel) AsCas12a. Data are shown as mean ± SD ( n = 3 independent experiments). ( C ) Comparison of the kinetic parameters of (left panel) FnCas12a-, (center panel) LbCas12a- and (right panel) AsCas12a-catalyzed pre-crRNA spacers cleavage with ssDNA or dsDNA activator. k cat / K m values are shown as mean ± SD ( n = 3 independent experiments). Statistical analysis was done by two tailed t -test.

Article Snippet: The protein-coding sequences of Francisella novicida U112 Cas12a (FnCas12a) (Addgene, Cat: 69976), Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) (Addgene, Cat: 69988), Acidaminococcus sp. Cas12a (AsCas12a) (Addgene, Cat: 69982), Cas12j (Addgene, Cat: 158801), Cas12i and Staphylococcus aureus Cas9 (SauCas9) (synthesized by SYNBIOB GENE) were separately subcloned into modified pET28a-His 6 -SUMO vector using a ClonExpress II One Step Cloning kit (Vazyme, Cat: C112-02).

Techniques: Activity Assay, Comparison, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: DNA target binding-induced pre-crRNA processing in type II and V CRISPR-Cas systems

doi: 10.1093/nar/gkae1241

Figure Lengend Snippet: Target binding-activated pre-crRNA spacer processing in vivo . ( A ) Schematic representation of an assay investigating the effect of target DNA on in vivo pre-crRNA spacer processing by FnCas12a or LbCas12a. ( B and C ) Northern blot analysis of the in vivo pre-crRNA processing activity of FnCas12a (panel B) and LbCas12a (panel C) in the presence or absence of target DNA. The RNA extracted from E. coli co-transformed with a plasmid encoding pre-crRNA, a plasmid containing or lacking target DNA and either the empty vector or overexpression vectors encoding WT Cas12a or its variants. All assays were repeated three times independently with similar results. ( D and E ) RNA-sequencing analysis of the in vivo pre-crRNA1 processing by FnCas12a (panel D) and pre-crRNA4 processing by LbCas12a (panel E) in the presence or absence of target DNA.

Article Snippet: The protein-coding sequences of Francisella novicida U112 Cas12a (FnCas12a) (Addgene, Cat: 69976), Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) (Addgene, Cat: 69988), Acidaminococcus sp. Cas12a (AsCas12a) (Addgene, Cat: 69982), Cas12j (Addgene, Cat: 158801), Cas12i and Staphylococcus aureus Cas9 (SauCas9) (synthesized by SYNBIOB GENE) were separately subcloned into modified pET28a-His 6 -SUMO vector using a ClonExpress II One Step Cloning kit (Vazyme, Cat: C112-02).

Techniques: Binding Assay, In Vivo, Northern Blot, Activity Assay, Transformation Assay, Plasmid Preparation, Over Expression, RNA Sequencing

Journal: Nucleic Acids Research

Article Title: DNA target binding-induced pre-crRNA processing in type II and V CRISPR-Cas systems

doi: 10.1093/nar/gkae1241

Figure Lengend Snippet: Cas12a and pre-crRNA based nucleic acid detection. ( A and B ) Schematic representation of mpox pseudovirus (panel A) and SARS-CoV-2 genome (panel B) detection. ( C ) Fluorescence real-time detection assay showing the sensitivity for mpox pseudovirus. ( D ) Comparison of net fluorescence intensity at 30 min using mpox pseudovirus with different copies. ( E ) Fluorescence real-time detection assay showing the sensitivity for SARS-CoV-2 genome. ( F ) Comparison of net fluorescence intensity at 30 min using SARS-CoV-2 genome with different copies. All data in panels (D) and (F) are shown as mean ± SD ( n = 3 independent experiments). Statistical analysis was done by two-tailed t -test.

Article Snippet: The protein-coding sequences of Francisella novicida U112 Cas12a (FnCas12a) (Addgene, Cat: 69976), Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) (Addgene, Cat: 69988), Acidaminococcus sp. Cas12a (AsCas12a) (Addgene, Cat: 69982), Cas12j (Addgene, Cat: 158801), Cas12i and Staphylococcus aureus Cas9 (SauCas9) (synthesized by SYNBIOB GENE) were separately subcloned into modified pET28a-His 6 -SUMO vector using a ClonExpress II One Step Cloning kit (Vazyme, Cat: C112-02).

Techniques: Fluorescence, Detection Assay, Comparison, Two Tailed Test