Targeted Modification of Mammalian DNA by a Novel Type V Cas12a Endonuclease from Ruminococcus bromii

Abstract

Type V Cas12a nucleases are DNA editors working in a wide temperature range and using expanded protospacer-adjacent motifs (PAMs). Though they are widely used, there is still a demand for discovering new ones. Here, we demonstrate a novel ortholog from Ruminococcus bromii sp. entitled RbCas12a, which is able to efficiently cleave target DNA templates, using the particularly high accessibility of PAM 5'-YYN and a relatively wide temperature range from 20 °C to 42 °C. In comparison to Acidaminococcus sp. (AsCas12a) nuclease, RbCas12a is capable of processing DNA more efficiently, and can be active upon being charged by spacer-only RNA at lower concentrations in vitro. We show that the human-optimized RbCas12a nuclease is also active in mammalian cells, and can be applied for efficient deletion incorporation into the human genome. Given the advantageous properties of RbCas12a, this enzyme shows potential for clinical and biotechnological applications within the field of genome editing.

Keywords: CRISPR; Cas endonuclease; genome editing; mammalian cells; site-directed mutagenesis.

Figure 1

Relationship of RbCas12a to other Cas12a orthologs. (A) Multiple sequence alignments of RbCpf1 with common orthologs of 18 Cas12a proteins that function in human cells. Catalytic Asp1194 and Glu1290 residues (numeration shown here for total alignment) are conserved (HkCas12a, Helcococcus kunzii ATCC 51366; CeCas12a, Coprococcus eutactus sp.; ErCas12a, Eubacterium rectale sp.; ArCas12a, Agathobacter rectalis strain 2789STDY5834884; LpCas12a, Lachnospira pectinoschiza strain 2789STDY5834886; EeCas12a, Lachnospira eligens ATCC 27750; AsCas12a, Acidaminococcus sp. BV36L; FnCas12a, Francisella novicida U112; TsCas12a, Thiomicrospira sp. XS5; Mb3Cas12a, Moraxella bovoculi sp.; Mb2Cas12a, Moraxella bovoculi AAX08_00205; MbCas12a, Moraxella bovoculi 237; RbCas12a, Ruminococcus bromii sp.; LbCas12a, Lachnospiraceae bacterium ND2006; PxCas12a, Pseudobutyrivibrio xylanivorans strain DSM 10317; PrCas12a, Pseudobutyrivibrio ruminis CF1b; BfCas12a, Butyrivibrio fibrisolvens MD2001; Lb2Cas12a, Lachnospiraceae bacterium MA2020; BsCas12a, Butyrivibrio sp. NC3005). (B) Neighbor-joining tree without distance corrections.

Figure 2

Effect of divalent cations on RbCas12a cleavage activity. Mean cleavage efficiencies and standard deviations calculated from three independent experiments of Me²⁺-dependent in vitro cleavage reactions are shown. Reactions in the same buffer but without any Me²⁺ addition served as controls.

Figure 3

Ruminococcus bromii CRISPR–Cas Type V system PAM sequence logo determined by linear DNA PAM library sequencing. (A) Schematic of the RbCas12a crRNA–DNA-targeting complex. The target DNA contains eight random nucleotides at the 5′-end PAM region. (B) Schematic illustration of the experimental assay used to discover the RbCas12a PAM position and identity. (C) Sequence logo for the determined RbCas12a PAM.

Figure 4

Investigation of RbCas12a PAM sequence by in vitro cleavage assay. The reactions contained 1.4 nM target DNA bearing the same protospacer but varying in the 5′-PAM sequence (as indicated above the panel), 346 nM recombinant RbCas12a, and 550 nM crRNA (a 0.1:35:55 nM ratio). Reactions were incubated for 30 min at 37 °C, and products were resolved by agarose gel electrophoresis. PAM frequency refers to the frequency of exact PAM in the cleaved PAM library. Mean cleavage efficiencies and standard deviations from three independent experiments are shown below the gel.

Figure 5

Effect of time on RbCas12a/crRNA and AsCas12a/crRNA cleavage activity. Target DNA cleavage by AsCas12a or RbCas12a programed with 500 nM crRNA at 1:3:30 target:Cas12a:RNA ratio. Mean cleavage efficiencies and standard deviations calculated from three independent experiments are shown.

Figure 6

Effect of split crRNA and spacer-only RNA on RbCas12a cleavage activity. (A) Target DNA cleavage by RbCas12a programed with 0.3 μM full-sized split or separate scaffold and spacer crRNA moieties at a 1:3:30 target:RbCas12a:RNA ratio. “Split” reactions contained both the scaffold and spacer RNAs. Mean cleavage efficiencies and standard deviations calculated from three independent experiments are shown. (B) Cleavage by RbCas12a programed with increasing concentrations (0.5, 1, 2, 2.5, and 5 μM of spacer RNA, corresponding to 1:5:50, 1:5:100, 1:5:200, 1:5:250, and 1:5:500 DNA:RbCas12a:crRNA ratios). Mean cleavage efficiencies and standard deviations calculated from three independent experiments are shown.

Figure 7

Thermofluor assay of RbCas12a. The left panel shows the melt curves, and the right panel shows the first derivative of the melt curves. The assay was performed with three independent replicas. The melt point of RbCas12a is 45.5 °C.

Figure 8

Effect of temperature on RbCas12a cleavage activity. Target DNA cleavage by RbCas12a programed with 500 nM crRNA at 1:3:30 target:Cas12a:RNA ratio. Mean cleavage efficiencies and standard deviations calculated from three independent experiments are shown.

Figure 9

Targeting the human DNMT1 gene with two crRNAs led to deletions. Top: Agarose gel visualization of PCR products after cell culture transfection with plasmids encoding RbCas12a and two crRNAs. Three independent experiments are shown as Rep1–3 on the second and fourth days of the experiment. Middle: A schematic representation of the PCR product with two guide RNAs highlighted. Bottom: Detailed maps with nucleotide sequences, created with SnapGene software.

Figure 10

Frequencies of break site flanking deletion. On the left side of the figure, a crRNA is indicated, which, complexed with RbCas12a, forms break sites flanking the left arm of the deletion. On the right side of the figure, the right crRNA is indicated. PAM sequences on both target and nontarget strands are highlighted in red font. The protospacer position on the target DNA strand and spacer moiety of crRNA is highlighted in blue font. Relative coverage depth in the edited region was calculated as depth coverage in certain positions divided by mean coverage depth outside of the edited region. DNA break efficiency after a certain position was assessed as the difference between relative depth in two neighboring positions.