Enhanced Cas12a editing in mammalian cells and zebrafish

Abstract

Type V CRISPR-Cas12a systems provide an alternate nuclease platform to Cas9, with potential advantages for specific genome editing applications. Here we describe improvements to the Cas12a system that facilitate efficient targeted mutagenesis in mammalian cells and zebrafish embryos. We show that engineered variants of Cas12a with two different nuclear localization sequences (NLS) on the C terminus provide increased editing efficiency in mammalian cells. Additionally, we find that pre-crRNAs comprising a full-length direct repeat (full-DR-crRNA) sequence with specific stem-loop G-C base substitutions exhibit increased editing efficiencies compared with the standard mature crRNA framework. Finally, we demonstrate in zebrafish embryos that the improved LbCas12a and FnoCas12a nucleases in combination with these modified crRNAs display high mutagenesis efficiencies and low toxicity when delivered as ribonucleoprotein complexes at high concentration. Together, these results define a set of enhanced Cas12a components with broad utility in vertebrate systems.

Figure 1.

Position and number of NLS improves genome editing by AsCas12a, LbCas12a and FnoCas12a. (A) General schematic of Cas12a (B). Schematic representation of a series of Cas12a constructs with different nuclear localization signals. Lesion rates determined by deep sequencing for SpCas9, AsCas12a, LbCas12a and FnoCas12a with different combinations of NLSs at the DNMT1 (C) and EMX1 (D) target sites, respectively. Boxed sequences represent SpCas9 targeting sites, red color labeled NGG PAM. Underlined sequences represent Cas12a targeting sites, blue color labeled TTTV PAM. Data are from three independent biological replicates performed on different days with expression constructs delivered by transient transfection in HEK293T cells (Supplementary Table S1). Error bars indicate ±s.e.m. Statistical significance is determined by two-tailed Student's t-test: ‘***’ denotes P < 0.001, ‘**’ denotes P < 0.01, respectively (Supplementary Table S7).

Figure 2.

Employing a full-length Direct-Repeat crRNA (DRf-crRNA) enhances editing efficiency. (A) General schematic of four different crRNAs: 19nt-truncated Direct-Repeat crRNA (DRt-crRNA), 35nt-full-length Direct-Repeat crRNA (DRf-crRNA), full-length Direct-Repeat crRNA with 3′ truncated Direct-Repeat (DRf-crRNA-DRt), full-length Direct-Repeat crRNA with 3′ full-length Direct-Repeat (DRf-crRNA-DRf). The 19-nt and 35-nt Direct Repeats are denoted with the dotted box, the guide sequences are marked in red. Scissors represents the Cas12a RNase domain, which process the crRNA at the RRS. The nuclease activities of each crRNA type are determined by deep sequencing for LbCas12a (B) and FnoCas12a (C) at 11 endogenous target sites on genome. Each box represents the 25th and 75th percentile and median is indicated by a line. Whiskers in the box plots are defined by the Tukey method. Statistical significance is determined by one-way analysis of variance (ANOVA), ‘****’ denotes P < 0.0001 (Supplementary Table S7). Deep sequencing data are from three independent biological replicates performed on different days with expression constructs delivered by transient transfection in HEK293T cells (Supplementary Table S2). Error bars indicate ± s.e.m. (D–G). The ability of Cas12a to process the Full-length Direct Repeat (DRf) is important for enhanced nuclease activity of this crRNA. Evaluation of the editing activity of Wild-type (WT) or RNase-dead Cas12a with different crRNA constructs (wild-type crRNA sequence or crRNA containing a mutation within the RRS sequence of crRNA) for LbCas12a (D, E) and FnoCas12a (F, G) at the DNMT1S3 (D, F) and EMX1S1 (E, G) target sites. Lesion rates determined by deep sequencing. Data are from three independent biological replicates performed on different days with expression constructs delivered by transient transfection in HEK293T cells (Supplementary Table S2). Error bars indicate ±s.e.m. Statistical significance is determined by two-tailed Student's t-test: ‘***’ denote P < 0.001, ‘**’ denote P < 0.01, ‘*’ denote P < 0.05, respectively (Supplementary Table S7).

Figure 3.

G-C swaps at specific position in the stem of the direct repeat increase editing efficiency. (A) General schematic of G–C swaps (indicated in red) at different position in the stem of the direct repeat hairpin. The activities of 9 different G-C swap crRNAs are determined by deep sequencing for LbCas12a (B) and FnoCas12a (C) at six different endogenous target sites on genome. Each box represents the 25th and 75th percentile and the middle line is the median. Whiskers in the box plots are defined by the Tukey method. Deep sequencing data are from three independent biological replicates performed on different days with expression constructs delivered by transient transfection in HEK293T cells (Supplementary Table S3). Error bars indicate ± s.e.m. Statistical significance is determined by one-way analysis of variance (ANOVA), ‘**’ and ‘***’ denote P < 0.01 and <0.001 respectively (Supplementary Table S7).

Figure 4.

Genome Editing by LbCas12a in zebrafish embryos. (A) Activity profiles of truncated Direct-Repeat crRNA (DRt-crRNA), full-length Direct-Repeat crRNA (DRf-crRNA) and G–C swapped crRNA (DRf-GC@13-crRNA) at four genomic sites in zebrafish embryos with and without heat shock using 4fmol RNP. Lesion rates are determined by deep sequencing. An aggregate analysis of the editing data (four target sites times three replicates) shows a significant increase in genome editing efficiency in fish embryos without heatshock (B) or with heatshock (C) for the DRf-crRNA and the DRf-GC@13-crRNA relative to the DRt-crRNA. (D) Activity profiles of three different crRNA frameworks at ten genomic sites in zebrafish embryos with and without heat shock treated with 24 fmol LbCas12a RNP. In the aggregate analysis across all ten target sites the DRf-GC@13-crRNA also provides a significant increase in genome editing efficiency in fish embryos without heatshock (E) or with heatshock (F). Deep sequencing data are from zebrafish embryos from three independent injections by three different individuals (Supplementary Table S5). For the bar charts, error bars indicate ± s.e.m. For each dot plot the three lines represent 75th, 50th and 25th percentile, respectively. Statistical significance is determined by one-way analysis of variance (ANOVA), ‘*’, ‘**’, ‘****’ denotes P values of <0.05, <0.01 and <0.0001, respectively (Supplementary Table S7).