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One-step strategy to create transgenic and KO mouse models (Easi-CRISPR) uses Megamer ssDNA donors and CRISPR RNPs

Quadros RM, Miura H, et al. (2017) Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol, DOI 10.1186/s13059-017-1220-4.

Read how the use of long, ssDNA donor sequences can improve HDR editing efficiency. These donor sequences, now available as custom Megamer Single-Stranded DNA Fragments from IDT, are injected into mouse embryos, along with CRISPR RNP complexes, to create transgenic and conditional knockout mouse models.

May 18, 2017

(Selected as a "Best of 2017" paper from Genome Biology.)

Background

Transgenic and conditional knockout mouse models are critical to genetic studies of gene function, yet exist for only ~25% of mouse genes. Creation of such models has been limited by the labor-intensive nature of homologous recombination methods that are required for embryonic stem cells. CRISPR-based homology-directed repair (HDR), using donor sequences of double-stranded DNA (dsDNA) or short, single-stranded oligodeoxynucleotides (ssODNs), has resulted in correctly targeted insertions. However, to date, these experiments have proven very inefficient, with the great majority (often >95%) of editing resulting in partial, duplicate, and off-target insertions, as well as deletions effected by non-homologous end joining (NHEJ). In the Quadros, et al. paper described here, a consortium of 7 research groups demonstrate improvements to genome editing efficiency in mouse zygotes by modifying CRISPR methods through the use of long, single-stranded DNA (ssDNA) donor sequences.

Experiments and discussion

Quadros et al. describe a novel single-step, genome editing strategy, Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), to deliver targeted genomic insertions at high frequency. The basis of the method is the microinjection of long, ssDNA donor sequences with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes.

High-fidelity, long, single-stranded DNA sequences have been previously unavailable. However, IDT now supplies Megamer® Single-Stranded DNA Fragments—custom, ssDNAs up to 2000 bases—that provide easy access to the ssDNA donor sequences needed for these experiments. Megamer Fragments were used here as donor DNAs. Synthetic crRNA and tracrRNA molecules were also supplied by IDT as Alt-R® CRISPR guide RNAs.

In their initial test case, the scientists targeted the mouse Pitx1 gene with a 1046-base donor sequence containing exon 2 flanked by LoxP sites (“floxed” exon 2; 862 bases) and homology arms of 91 and 93 bases. The donor sequence and ctRNP were injected into mouse zygotes to effect HDR. The resulting 10 live offspring were genotyped using PCR assays specific to the targeted insertion of each LoxP site and the entire Pitx1 floxed exon. 40% of the progeny had the desired insertion.

To demonstrate the reproducibility of the method, the research groups targeted an additional 6 mouse genes (Ambra1, Col12a1, Ubr5, Syt1, Syt9, and Ppp2r2a) with similarly designed floxed donor sequences. Genotyping of the 7 genes targeted with floxed donor sequences showed that 43% of resulting mouse progeny contained at least 1 correctly targeted allele, with efficiencies that ranged between 8.5 and 100% across the different loci.

In an additional series of experiments, Quadros et al. showed that the Easi-CRISPR method can also be used to create knock-in (KI) alleles with high efficiency. The groups targeted 6 mouse genes with ssDNA donors of 0.8–1.4 kb, containing homology arms and sequences encoding either FlpO recombinase, the reverse tetracycline transactivator (rtTA), or the reporters mCherry and mCitrine, along with the appropriate guide RNAs. Analysis of PCR genotyping data from the offspring resulting from the injected zygotes yielded targeted insertion efficiencies of 25–67%.

The Easi-CRISPR method can provide a robust, simplified process for creating conditional and targeted insertion alleles, demonstrated by the successful targeting of 13 loci, with efficiencies well above those cited in previous studies. The ready availability of custom, synthetic, long ssDNAs (Megamer Single-Stranded DNA Fragments) and guide RNAs (Alt-R CRISPR Guide RNAs) further facilitate quick and precise animal genome engineering.

Definitions

Floxed sequence

A DNA sequence flanked by LoxP sites, making it susceptible to recombination catalyzed by Cre recombinase.

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