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Electroporation of Mice Zygotes With Dual Guide RNA/Cas9 Complexes for Simple and Efficient Cloning-Free Genome Editing

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Electroporation of Mice Zygotes With Dual Guide RNA/Cas9 Complexes for Simple and Efficient Cloning-Free Genome Editing

Marie Teixeira et al. Sci Rep.

Erratum in

Abstract

In this report, we present an improved protocol for CRISPR/Cas9 genome editing in mice. The procedure consists in the electroporation of intact mouse zygotes with ribonucleoprotein complexes prepared in vitro from recombinant Cas9 nuclease and synthetic dual guide RNA. This simple cloning-free method proves to be extremely efficient for the generation of indels and small deletions by non-homologous end joining, and for the generation of specific point mutations by homology-directed repair. The procedure, which avoids DNA construction, in vitro transcription and oocyte microinjection, greatly simplifies genome editing in mice.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
General structure of the dual guide RNA. Two synthetic RNA molecules are annealed. The short variable 42mer called crRNA has its first 20 nt designed to match a genomic sequence located immediately upstream to a PAM consensus motif (5′NGG) and the sequence is identical to the bottom genomic DNA strand on this figure. The second RNA, called tracrRNA, is a constant 72mer with partial homology to the crRNA, which secondary structure permits the tight association to the Cas9 nuclease (grey). Black crosses indicate the modified nucleotides, to which 2′-O-methyl and 3′ phosphorothioate are added to protect from nuclease degradation. Arrows indicate the cutting sites of the Cas9 nuclease.
Figure 2
Figure 2
Mice genotyping (A) Use of a dual guide RNA to generate short indels on Brcc3 gene. DNA Sanger sequencing of control and mutated F0 n°4 mice are shown. Top chromatogram represents a part of wild-type Brcc3 gene: guide sequence is underlined in blue, green rectangle indicates PAM, red arrow indicates Cas9 cutting site. Bottom chromatogram represents mutant mouse n°4, in which NHEJ generated a 2 nt deletion (dotted red lines) without apparent mosaicism, as correlated by TIDE software analysis represented on the right side of chromatograms. (B) Simultaneous use of two dual guide RNAs to generate short indels on two vasohibin genes located on different chromosomes. DNA Sanger sequencing of control and mutated F0 mice n°2 are shown for Vash1 (top chromatograms) and Vash2 (bottom). The TIDE software is used to analyze the spectrum and frequency of indels, as represented on the right side of chromatograms. On control sequences, guide sequences are underlined in blue and green rectangles indicate PAM. Red arrows indicate Cas9 cutting site. On Vash1 mutant lane, mosaicism and heterozygosity generate a scrambled sequence. Vash2 mutant chromatogram represents a case without apparent mosaicism, in which NHEJ generated a 28 nt deletion (dotted red lines) on both Vash2 alleles. (C) Simultaneous use of two dual guide RNAs to generate a 83 bp deletion in Ctse gene. Agarose gel analysis (cropped picture) of PCR performed on F0 mice genomic DNA, showing the frequent presence of a shorter PCR product of the expected size: the intended deletion of 83 bp leaves 398 bp instead of the 481 bp for the wild-type allele. Although most mice seem to harbor the expected deletion, additional small indels were identified by Sanger sequencing for some of them. Samples 8, 9 and 12 have larger deletion. Samples 10 and 17 repeatedly failed to amplify, suggesting large deletion, as DNA integrity was verified with primers targeting an independent locus (Supplementary Fig. S3). C represents control mouse with only the 481 bp wild-type allele. Full-length gels are presented in Supplementary Fig. S3. (D) Generation of specific point mutations using ssODN as template for HDR. A fraction of the Mct8 genomic sequence and the corresponding reading frame are shown. The sequence underlined in blue is complementary to the 5′ end of the crRNA. The green rectangle indicates the complement of the PAM sequence. Red arrow indicates Cas9 cutting site. The ssODN has perfect homology for the Mct8 gene and is identical to the “non PAM” strand on 92 nt at the 5′ end and 36 nt at the 3′ end. Mismatches are with red letters. The first two mismatches change the proline codon into a leucine codon. The following ones eliminate the PAM sequence, preventing Cas9 cleavage after HDR, and create a Pac I restriction site facilitating mouse genotyping. Top chromatogram represents control mouse sequence and the bottom one represents mutated F0 male n°7. Asterisks indicate mutated nucleotides.

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