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Lb2Cas12a efficiently cleaved mammalian genomes at both integrated and endogenous targets (A) A phylogenetic tree generated by Phylo.io based on an alignment of Cas12a orthologs of the indicated species. , (B) A representation of the indicated Cas12a orthologs with domains indicated. WED: crRNA binding and processing domains, split by REC and PI domains into WED-I, II, and III (green). REC: DNA binding domains REC1 and REC2 (gray). PI: PAM-interacting domain (orange). RuvC: DNA cleavage domains, split by BH and NUC domains into RuvC-I, II, and III (blue). BH: bridge helix (light green). Nuc: DNA processing domain (red). Number at right indicates the number of amino acids of each ortholog. (C) A diagram illustrating the double-stranded-break-induced gain-of-expression assay used in subsequent figures. A construct with an in-frame (+1) crRNA target sequence (orange) preceding an out-of-frame (+3) luciferase gene (light green) is stably integrated into the genomes of HEK293T cells. Upon cleavage by a Cas12a/crRNA complex, non-homologous end-joining (NHEJ) repair places approximately one-third of the downstream luciferase genes in frame (dark green), enabling their expression. Luciferase activity reflects the efficiency of Cas12a-mediated cleavage. (D) A list of target sequences used in subsequent panels with their preceding PAMs (blue). (E) The editing efficiency of <t>AsCas12a,</t> LbCas12a, and Lb2Cas12a, measured with the assay shown in panel (C), were compared using the six indicated lentivirally integrated targets. The means of three independent replicates are shown, with error bars indicating ± standard error of the mean (SEM). The significance of differences with Lb2Cas12a are indicated above bars representing AsCas12a and LbCas12 (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; values of p > 0.05 are indicated in the graph), as determined by two-way ANOVA, followed by Tukey’s multiple comparison tests. (F) Lb2Cas12a edited an endogenous locus of the DNMT1 gene (DNMT1-3) with high efficiency. A T7E1 assay was used to compare the editing efficiencies of the Cas12a orthologs. Percent edited, indicated beneath the figure, is calculated as in Guschin et al. and indicates the average of three independent biological replicates. Neg: negative-control cells transfected with vector alone. (G) Expression of AsCas12a, LbCas12a, and Lb2Cas12a in HEK293T cells used in (F). A western blot, representative of three independent biological replicates with similar results, is shown.
Wild Type Ascas12a, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Lb2Cas12a efficiently cleaved mammalian genomes at both integrated and endogenous targets (A) A phylogenetic tree generated by Phylo.io based on an alignment of Cas12a orthologs of the indicated species. , (B) A representation of the indicated Cas12a orthologs with domains indicated. WED: crRNA binding and processing domains, split by REC and PI domains into WED-I, II, and III (green). REC: DNA binding domains REC1 and REC2 (gray). PI: PAM-interacting domain (orange). RuvC: DNA cleavage domains, split by BH and NUC domains into RuvC-I, II, and III (blue). BH: bridge helix (light green). Nuc: DNA processing domain (red). Number at right indicates the number of amino acids of each ortholog. (C) A diagram illustrating the double-stranded-break-induced gain-of-expression assay used in subsequent figures. A construct with an in-frame (+1) crRNA target sequence (orange) preceding an out-of-frame (+3) luciferase gene (light green) is stably integrated into the genomes of HEK293T cells. Upon cleavage by a Cas12a/crRNA complex, non-homologous end-joining (NHEJ) repair places approximately one-third of the downstream luciferase genes in frame (dark green), enabling their expression. Luciferase activity reflects the efficiency of Cas12a-mediated cleavage. (D) A list of target sequences used in subsequent panels with their preceding PAMs (blue). (E) The editing efficiency of AsCas12a, LbCas12a, and Lb2Cas12a, measured with the assay shown in panel (C), were compared using the six indicated lentivirally integrated targets. The means of three independent replicates are shown, with error bars indicating ± standard error of the mean (SEM). The significance of differences with Lb2Cas12a are indicated above bars representing AsCas12a and LbCas12 (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; values of p > 0.05 are indicated in the graph), as determined by two-way ANOVA, followed by Tukey’s multiple comparison tests. (F) Lb2Cas12a edited an endogenous locus of the DNMT1 gene (DNMT1-3) with high efficiency. A T7E1 assay was used to compare the editing efficiencies of the Cas12a orthologs. Percent edited, indicated beneath the figure, is calculated as in Guschin et al. and indicates the average of three independent biological replicates. Neg: negative-control cells transfected with vector alone. (G) Expression of AsCas12a, LbCas12a, and Lb2Cas12a in HEK293T cells used in (F). A western blot, representative of three independent biological replicates with similar results, is shown.

Journal: Molecular Therapy. Nucleic Acids

Article Title: A more efficient CRISPR-Cas12a variant derived from Lachnospiraceae bacterium MA2020

doi: 10.1016/j.omtn.2021.02.012

Figure Lengend Snippet: Lb2Cas12a efficiently cleaved mammalian genomes at both integrated and endogenous targets (A) A phylogenetic tree generated by Phylo.io based on an alignment of Cas12a orthologs of the indicated species. , (B) A representation of the indicated Cas12a orthologs with domains indicated. WED: crRNA binding and processing domains, split by REC and PI domains into WED-I, II, and III (green). REC: DNA binding domains REC1 and REC2 (gray). PI: PAM-interacting domain (orange). RuvC: DNA cleavage domains, split by BH and NUC domains into RuvC-I, II, and III (blue). BH: bridge helix (light green). Nuc: DNA processing domain (red). Number at right indicates the number of amino acids of each ortholog. (C) A diagram illustrating the double-stranded-break-induced gain-of-expression assay used in subsequent figures. A construct with an in-frame (+1) crRNA target sequence (orange) preceding an out-of-frame (+3) luciferase gene (light green) is stably integrated into the genomes of HEK293T cells. Upon cleavage by a Cas12a/crRNA complex, non-homologous end-joining (NHEJ) repair places approximately one-third of the downstream luciferase genes in frame (dark green), enabling their expression. Luciferase activity reflects the efficiency of Cas12a-mediated cleavage. (D) A list of target sequences used in subsequent panels with their preceding PAMs (blue). (E) The editing efficiency of AsCas12a, LbCas12a, and Lb2Cas12a, measured with the assay shown in panel (C), were compared using the six indicated lentivirally integrated targets. The means of three independent replicates are shown, with error bars indicating ± standard error of the mean (SEM). The significance of differences with Lb2Cas12a are indicated above bars representing AsCas12a and LbCas12 (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; values of p > 0.05 are indicated in the graph), as determined by two-way ANOVA, followed by Tukey’s multiple comparison tests. (F) Lb2Cas12a edited an endogenous locus of the DNMT1 gene (DNMT1-3) with high efficiency. A T7E1 assay was used to compare the editing efficiencies of the Cas12a orthologs. Percent edited, indicated beneath the figure, is calculated as in Guschin et al. and indicates the average of three independent biological replicates. Neg: negative-control cells transfected with vector alone. (G) Expression of AsCas12a, LbCas12a, and Lb2Cas12a in HEK293T cells used in (F). A western blot, representative of three independent biological replicates with similar results, is shown.

Article Snippet: Wild-type AsCas12a (pcDNA3.1-hAsCpf1), LbCas12a (pcDNA3.1-hLbCpf1), and Lb2Cas12a (pcDNA3.1-hLb2Cpf1) and pX330 (pX330-U6-chimeric_BB-CBh-hSpCas9) 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 Cas12a variants in E. coli for protein production.

Techniques: Generated, Binding Assay, Expressing, Construct, Sequencing, Luciferase, Stable Transfection, Non-Homologous End Joining, Activity Assay, Comparison, Negative Control, Transfection, Plasmid Preparation, Western Blot