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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.

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.

Definition

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

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.

Product focus—genome editing with long ssDNA and Alt-R® CRISPR Reagents

Megamer™ Single-Stranded DNA Fragments

These custom-designed, sequence-verified, single-stranded DNAs range in length from 201–2000 bases. Use them in applications such as homology-directed repair (HDR), in research using CRISPR-mediated genome editing, in vitro transcription, and more. Synthesized with clonally purified DNA, they offer the greatest purity available.

Learn more about Megamer Single-Stranded DNA Fragments.


Alt-R CRISPR-Cas9 System

The Alt-R CRISPR-Cas9 System includes all the reagents needed for successful genome editing. Based on the natural S. pyogenes CRISPR-Cas9 system, the Alt-R CRISPR-Cas9 System offers numerous advantages over alternative methods:

  • Higher on-target potency than other CRISPR systems
  • Precision control with delivery of Cas9 ribonucleoprotein (RNP)
  • Efficient delivery of the RNP with lipofection, electroporation, or microinjection
  • No toxicity or innate immune response activation, in contrast to in vitro transcribed Cas9 mRNA and sgRNAs

Learn more about the Alt-R CRISPR-Cas9 System.


Alt-R CRISPR-Cpf1 System

The Alt-R CRISPR-Cpf1 System recognizes an AT-rich PAM site, providing CRISPR target sites that are not available with the CRISPR-Cas9 System. In addition, Cpf1 nuclease produces a staggered cut with a 5′ overhang. These reagents:

  • Enable genome editing in organisms with AT-rich genomes
  • Allow interrogation of additional genomic regions compared to Cas9
  • Require simply complexing the crRNA with the Cpf1 protein—no tracrRNA needed
  • Permit efficient delivery of the RNP into cells by electroporation or microinjection

Learn more about the Alt-R CRISPR-Cpf1 System.


CRISPR support reagents

Additional CRISPR reagents extend the ease-of-use and performance of the Alt-R system through options for fluorescent visualization, enhanced nuclease transfection, and genome editing detection.

Find out more about IDT’s entire line of CRISPR products.


Related reading

Using CRISPR genome editing for gene knockout and homology-directed repair (HDR)—Webinar review: Watch our webinar recording for expert guidance on a complete CRISPR genome editing workflow, including available tools and protocols. Also, see what we have learned about homology-directed repair and a new option for repair templates.

Successful CRISPR genome editing in hard-to-transfect cells (i.e., Jurkat cells)—Use the conditions presented here for Clone E6-1 Jurkat cells as a starting point for optimization of CRISPR reagent delivery in cell types requiring electroporation.

CRISPR genome editing: 5 considerations for target site selection—Learn how your genome editing experiments can be improved with 5 quick tips for target selection and with reagents from the Alt-R® CRISPR-Cas9 System.


Review other DECODED Online newsletter articles on CRISPR in genome editing applications.


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Author: Ellen Prediger, PhD, is a scientific writer at IDT.

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