ErCas12a CRISPR-MAD7 for Model Generation in Human Cells, Mice, and Rats

Abstract

MAD7 is an engineered class 2 type V-A CRISPR-Cas (Cas12a/Cpf1) system isolated from Eubacterium rectale. Analogous to Cas9, it is an RNA-guided nuclease with demonstrated gene editing activity in Escherichia coli and yeast cells. Here, we report that MAD7 is capable of generating indels and fluorescent gene tagging of endogenous genes in human HCT116 and U2OS cancer cell lines, respectively. In addition, MAD7 is highly proficient in generating indels, small DNA insertions (23 bases), and larger integrations ranging from 1 to 14 kb in size in mouse and rat embryos, resulting in live-born transgenic animals. Due to the different protospacer adjacent motif requirement, small-guide RNA, and highly efficient targeted gene disruption and insertions, MAD7 can expand the CRISPR toolbox for genome enginnering across different systems and model organisms.

FIG. 1.

A DNA mismatch detection assay using T7EI enzyme for non-overlapping MAD7 and Cas9 targets using expressed enzyme and guide RNAs (gRNAs) in HCT116 cells. MAD7 or Cas9 enzyme and corresponding gRNAs to gene target PPIB (A) or DNMT3B (B) listed above each gel. Polymerase chain reaction (PCR) amplicons for each gene editing target site were produced from cell lysate generated from each individual transfection. PCR amplicons from untransfected cells for each gRNA target site were used as negative controls (Neg. Con.) in the T7EI assay. Percent indel formation is shown at the bottom of the gels plus or minus standard deviation.

FIG. 2.

Enhanced green fluorescent protein (eGFP) N-terminal tagging of the human CBX1 gene. (A) Four MAD7 gRNAs (red) and a Cas9 gRNA (blue) each designed to direct cleavage of the genomic gene target around the start codon of CBX1 denoted by the black arrow. (B) U2OS cells co-transfected with plasmids expressing either MAD7 or Cas9 nuclease and the corresponding gRNA, with a repair plasmid to direct homology-directed repair–mediated insertion of the eGFP gene. eGFP expression was detected via epifluorescence microscopy.

FIG. 3.

Mouse Rosa26 target with oligo donor integrations. (A) Schematic representation of the Rosa26 locus and the targeting gRNA. Dark bars represent exons and the solid line intron 1. The blue text represents the protospacer adjacent motif site and the remaining targeting sequence. The size of the homology arms (HA; bases) within the oligo donor is listed and the lines represent where it starts within the target region. The bottom schematic represents the HA (R, right; L, left) in yellow and the T7-BamHI integration sequence in orange. (B) PCR amplification products that flank the Rosa26 locus from purified DNA of isolated oligo-donor micro-injected embryonic day 14 (E14) embryos (left panel), BamHI restriction digests of the amplification products (right panel), and PCR amplification products for live-born pups (bottom panel). The dark arrow on the right of each gel represents the expected size (302 bp) of the PCR amplification product. The numbers to the left represent the location of the DNA size markers in base pairs.

FIG. 4.

Mouse Rosa26 target with large plasmid donor integrations. (A) Schematic representation of the Rosa26 locus, targeting gRNA and the large targeting donor plasmid. The size of the HA within the plasmid donor and the lines to where they start within the target region. The middle 66 bp listed represents the distance between where the HA start within the Rosa26 locus. The bottom schematic represents the HA (R, right; L, left) in yellow, CAG promoter in blue, poly(A) signal in black, and the remaining Cas9-eYFP-Cpf1 gene fusion. (B) Upstream (top panel) and downstream (bottom panel) junction PCR amplification products from purified DNA from wild-type (WT) and founder mice. The numbers on the right in each panel represent DNA size markers in base pairs.

FIG. 5.

Cre targeting of the Rat Calb2 gene. (A) Schematic representation of the Calb2 locus, targeting gRNA and the Cre-targeting donor plasmid. The locations of start (ATG) and stop (TAA) codons are listed above and the dotted lines to where the targeting vector will integrate upstream of the stop codon. The Calb2 locus is shortened for simplicity and to include the relevant exons for Cre-targeting. The size of the HA within the plasmid donor and the lines to where they start within the target region. The bottom schematic represents the HA (R, right; L, left) in yellow and the Cre gene in orange. (B) Upstream (top panel) and downstream (bottom panel) junction PCR amplification products from purified DNA from WT and founder rats. The numbers on the right in each panel represent DNA size markers in base pairs.