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. 2018 Nov 27;4:63.
doi: 10.1038/s41421-018-0069-3. eCollection 2018.

Repurposing CRISPR-Cas12b for mammalian genome engineering

Fei Teng #  1   2   3 , Tongtong Cui #  1   2   3 , Guihai Feng  1   2 , Lu Guo  1   2   3 , Kai Xu  1   2   3 , Qingqin Gao  1   2   3 , Tianda Li  1   2 , Jing Li  1   2 , Qi Zhou  1   2   3 , Wei Li  1   2   3
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Repurposing CRISPR-Cas12b for mammalian genome engineering

Fei Teng et al. Cell Discov. .
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Abstract

The prokaryotic CRISPR-Cas adaptive immune systems provide valuable resources to develop genome editing tools, such as CRISPR-Cas9 and CRISPR-Cas12a/Cpf1. Recently, CRISPR-Cas12b/C2c1, a distinct type V-B system, has been characterized as a dual-RNA-guided DNA endonuclease system. Though being active in vitro, its cleavage activity at endogenous genome remains to be explored. Furthermore, the optimal cleavage temperature of the reported Cas12b orthologs is higher than 40 °C, which is unsuitable for mammalian applications. Here, we report the identification of a Cas12b system from the Alicyclobacillus acidiphilus (AaCas12b), which maintains optimal nuclease activity over a wide temperature range (31 °C-59 °C). AaCas12b can be repurposed to engineer mammalian genomes for versatile applications, including single and multiplex genome editing, gene activation, and generation of gene mutant mouse models. Moreover, whole-genome sequencing reveals high specificity and minimal off-target effects of AaCas12b-meditated genome editing. Our findings establish CRISPR-Cas12b as a versatile tool for mammalian genome engineering.

Conflict of interest statement

A patent application has been filed relating to this work. The authors declare that there is no conflict of financial interests, and they plan to deposit the reagents in Addgene to freely share with the academic community.

Figures

Fig. 1. Cas12b nucleases cleave DNA in vitro guided by crRNA/tracrRNA.
a Schematic illustration of the genomic architecture of CRISPR-Cas12b from Alicyclobacillus acidiphilus (NBRC 100859) (left) and the crRNA/tracrRNA duplex (right). b In vitro cleavage activity of AaCas12b at various temperatures. The cleavage rate is shown under the cleaved lanes. c In vitro validation of the PAM requirements of AaCas12b showing that PAMs matching the 5′-TTN sequence can be efficiently cleaved. The cleavage rate is shown under the cleaved lanes. d Cleavage site determination of AaCas12b by sequencing the cleavage products. The cleavage sites are indicated by red triangles in the left panel. TS target strand, NTS non-target strand
Fig. 2. Genome editing by Cas12b nucleases in human 293FT cells.
a Schematic illustration of the eukaryotic expression strategy of the Cas12b and its cognate guide RNA (gRNA). The pre-tRNAGly, an endogenous RNA processing system for cleavage of transcripts, was hijacked to simultaneously express the tracrRNA and crRNA using a single human U6 promoter. b Schematic illustration of the human RNF2 target site 1 and crRNA/tracrRNA duplex. Red letters indicate the PAM sequences. c T7EI analysis of the indels produced by Cas12b orthologs (AaCas12b, AkCas12b, AmCas12b, and BsCas12b) at the human RNF2 target site 1. The indel rate is shown under the lanes with mutation. mock, an U6 empty vector without crRNA/tracrRNA expression. GFP, an empty backbone vector without Cas12b protein expression. d Sanger sequencing results showing the indels in human RNF2 target site 1 produced by AaCas12b and AkCas12b. Blue dashes, deleted bases; purple lowercases, insertions or mutations; red uppercases, PAM. e Effects of human plasma incubation on the nuclease activity of SpCas9 and AaCas12b. After incubation in human plasma at indicated concentrations for 12 h at 37 °C, in vitro DNA cleavage assay was conducted. The cleavage rate is shown under the cleaved lanes
Fig. 3. Engineering of the AaCas12b system for multiplex mammalian genome editing.
a Schematic illustration of the sequences and structure of chimeric single guide (sgRNA) (top), and depiction of the truncation strategy of the sgRNA scaffold (bottom). b Effects of truncation of stem-loop 1 within the sgRNA scaffold on the nuclease activity of AaCas12b in human cells. The indel rate is shown under the lanes with mutation. c Effects of truncation of stem-loop 2 and stem-loop 3 within the sgRNA scaffold on the nuclease activity of AaCas12b in human cells. The indel rate is shown under the lanes with mutation. d AaCas12b facilitates multiplex genome editing by simultaneously targeting CCR5, CD34, DNMT1, and RNF2, using four individual sgRNAs containing spacers. (Top) Schematic illustration of the target sites in the human genome. (Bottom) All the four sgRNAs mediate efficient protospacer cleavage. The indel rate is shown under the lanes with mutation
Fig. 4. dAaCas12b-medaited gene activation in human cells.
a Schematic illustration of AaCas12b domain structure showing the positions of catalytic residue mutations. The catalytic residues were identified based on sequence homology of AaCas12b and A. acidoterrestris Cas12b (AacCas12b) (PDB: 5WQE). b In vitro DNA cleavage analysis of mutation of indicated nuclease mutant or wild-type (WT) AaCas12b proteins. The cleavage rate is shown under the cleaved lanes. c T7EI analysis showing effects of mutation of AaCas12b catalytic residues on DNA targeting in 293FT cells. The indel rate is shown under the lanes with mutation. GFP, an empty backbone vector without Cas12b protein expression. d Schematic illustration of sgRNA scaffold-based recruitment enabling simultaneous activation of independent target genes. The sgRNA construct with MS2 RNA hairpin recruits MCP-VP64 or MCP-VP64-p65-Rta (VPR) to activate endogenous gene expression in human 293FT cells. e The sgRNA scaffold recruits MCP-VPR or MCP-VP64 to simultaneously activate endogenous expression of IL1B and HBG1 in human 293FT cells combined with dAaCas12b or dSpCas9 expression, respectively. Cells transfected with only empty vectors were used as control (Ctrl)
Fig. 5. AaCas12b-mediated genome editing in mice.
a Schematic illustration of AaCas12b sgRNA targeting exon 3 of the mouse Nrl gene. The PAM and target sequences are colored in red and blue, respectively. The cleavage sites are indicated with red triangles. b Schematic illustration of AaCas12b RNP delivery into 1-cell stage mouse embryos by microinjection. c Summary of the mutants generated via AaCas12b RNP microinjection with sgRNA targeting the Nrl and Prmt7 genes, respectively. d T7EI-based genotyping assay identifying founder mice derived from embryos injected with AaCas12b RNPs targeting the mouse Nrl gene. The numbers in red denote newborn mice with induced indel mutations. The indel rate is shown under the lanes with mutation. e Mutated Nrl alleles observed in the founder mice in Fig. 5d. Blue dashes, deleted bases; purple lowercases, insertions or mutations; red uppercases, PAM. Indel frequencies are indicated
Fig. 6. Cleavage specificity and off-target effects of AaCas12b in mammalian genomes.
a Analysis of cleavage specificity of AaCas12b/sgRNA in human and mouse cells using sgRNAs carrying single base-pair mismatches in the guide sequence. Error bars indicate standard errors of the mean (s.e.m.), n = 3. b (Top) Schematic illustration of AaCas12b target sites in the human CCR5 and RNF2 loci, respectively. (Bottom) Indel frequencies induced by AaCas12b directed by sgRNAs targeting endogenous CCR5 and RNF2 sites and their corresponding off-target sites in human 293FT cells. Mutation frequencies were assessed by T7EI assay. Error bars indicate s.e.m., n = 2. c WGS analysis of genomic DNAs of RNF2-targeted 293FT cells. None of 2598 (RNF2-site 1) and 3394 (RNF2-site 2) sites identified with Cas-OFFinder in the reference genome (hg38) that differed from the on-target site by up to five mismatches harbored indels in the mutated genome. d Whole-genome sequencing (WGS) analysis of genomic DNAs of Nrl-mutated mouse gonads. None of the 2657 sites identified using Cas-OFFinder in the reference genome (mm10) that differed from the on-target site by up to five mismatches harbored indels in the mutated genome
Fig. 7. Off-target effects induced by AaCas12b, AsCas12a and SpCas9 in mammalian genomes.
a (Left) Schematic showing the targeting sequences of AaCas12b, AsCas12a and SpCas9 in mouse Nrl locus. (Right) Activities of AaCas12b, AsCas12a and SpCas9 targeted to mouse Nrl locus using respective guide RNAs with single mismatches in mouse ES cells. Mutation frequencies were assessed by T7EI assay. Error bars indicate standard errors of the mean (s.e.m.), n = 3. b (Upper) Schematic showing AaCas12b and SpCas9 targeting sites in the human RNF2 locus. (Lower) Frequencies of induced indels induced at on- and off-target sites by AaCas12b and SpCas9 in human 293FT cells. Mutation frequencies were assessed by T7EI assay. Error bars indicate s.e.m., n = 2. c Whole-genome sequencing (WGS) analysis potential off-target sites with one to five mismatches to gRNAs induced by AaCas12b or SpCas9 in the human genome and the amount of mutated sites observed by WGS
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