Easicrispr Efficient Germline Modification With Long Ssdna Donors

. 2018 Jan;13(1):195-215.

doi: 10.1038/nprot.2017.153. Epub 2017 Dec 21.

Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors

Affiliations

• PMID: 29266098
• PMCID: PMC6058056
• DOI: 10.1038/nprot.2017.153

Free PMC article

Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors

Hiromi Miura  et al. Nat Protoc. 2018 Jan .

Free PMC article

Abstract

CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research involve knock-in (reporters or recombinases) or gene replacement (e.g., conditional knockout alleles containing exons flanked by LoxP sites). A few methods for creating such models have been reported that use double-stranded DNA as donors, but their efficiency is typically 1-10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement. We call this method efficient additions with ssDNA inserts-CRISPR (Easi-CRISPR) because it is a highly efficient technology (efficiency is typically 30-60% and reaches as high as 100% in some cases). The protocol takes ∼2 months to generate the founder mice.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

C.B.G., M.O. and H.M. have filed a patent application relating to the work described in this manuscript on international application number PCT/US2016/035660 filed June 3, 2016 (DNA editing using single stranded DNA).

Figures

Figure 1:. Schematic of Easi-CRISPR.

The procedure involves three broad stages: (i) assembling of CRISPR Ribonucleoprotein components (crRNA + tracrRNA + Cas9 Protein: ctRNP) and generating a long ssDNA donor (Steps 1–40); (ii) preparation of Easi-CRISPR components, their microinjection into mouse zygotes and generation of founder offspring (Steps 41–53), and; (iii) genotyping of offspring (Steps 54–59). All experimental procedures using animals should be carried out according to relevant institutional regulations for animal usage.

Figure 2:. Design principles of knocking-in using Easi-CRISPR and the architecture of ssDNA donor.

(a) Genomic locus of a hypothetical gene's last exon showing the stop codon in red (TGA). The green sequence upstream of- and the blue sequence downstream of- the stop codon will be included as parts of upstream and downstream arms of the ssDNA donor, respectively. (b) Hypothetical guide search results showing four guide options along with their protospacer adjacent motif (PAM) sequences (5'-NGG-3'). The guide that cleaves immediately upstream of the stop codon will be the most preferred guide for use in the Easi-CRISPR procedure. If such a guide is not available for a given locus, a next closest one should be chosen. In the example shown here, a guide that cuts 7 bases downstream should be the second option. The guide option # 3 and "the least preferred guide" cleave at −16 bases and +19 bases from the target site. Farther the guide from the target site, poorer will be the correct insertion frequency because imprecise insertion rates become higher. If either of the last two guides are chosen, the donor cassette should preferably contain mutation/s in the guide recognition sites (or PAM) to prevent Cas9 re-cleaving after the cassette is inserted. (c) Schematic of a donor DNA showing T7 promoter and the ssDNA region. The T7 promoter sequence is included in the dsDNA template (used for ivTRT) but it will not get included in the final ssDNA. Green arrow shows the primer for reverse transcription. (d) Knock-in locus showing correct fusion of new sequence.

Figure 3:. Design principles of floxing using Easi-CRISPR and the architecture of ssDNA donor.

(a) Genomic locus of a hypothetical gene's target exon and its surrounding regions, used for guide search. Hypothetical guide search results showing multiple guide options for left and right guides. Two guides (one each for upstream and downstream ends) with high guide scores, and least (or no) off target cleavage sites, are chosen. The typical distance between the two guide target sites are ~0.5 kb to 0.8 kb, and they should be placed sufficiently away from the target exon (at least about 100 bases), to prevent disruption of splice donor/acceptor regulatory elements in the floxed allele. (b) Schematic of donor DNA showing T7 promoter (as part of dsDNA template) and the actual part of ssDNA donor. The LoxP insertion sites are placed precisely at the Cas9 cleavage sites. Green arrow shows the primer for reverse transcription. (c) Floxed locus showing correct insertion of the new sequence.

Figure 4:. Design principles of inserting knock-down cassette using Easi-CRISPR and the architecture of ssDNA donor.

(a) Genomic locus of a hypothetical gene's target intron and its surrounding regions chosen for guide search. The guide search results showing multiple guide options. A guide with high score, and least (or no) off target cleavage sites, is chosen for the design. (b) Schematic of a donor DNA showing T7 promoter and the ssDNA region. The T7 promoter sequence is included in the dsDNA template (used for ivTRT) but it will get excluded in the final ssDNA. Green arrow shows the primer for reverse transcription. (d) Knock-in locus showing correct insertion of the new sequence.

Figure 5:. Schematic of ivTRT and ssDNA preparation steps.

A dsDNA template can be a PCR product, or a plasmid with a suitable restriction enzyme (RE) site distal to the insertion cassette (Steps 1–2). The gel on the right shows a plasmid digested with a RE. RNA is synthesized using in vitro transcription (Steps 3–18). The gel on the right shows an RNA of ~ 900 bases long. cDNA is synthesized by reverse transcription using a reverse primer (Steps 19–23). The gel on the right shows a sample ssDNA (cDNA). Note that the cDNA preparation typically runs like a smear with a prominent band within the smear. Purification of ssDNA (Steps 24–35). The image on the left shows the gel after excision of the prominent band (for purification) and the gel on the right shows the purified ssDNA. The GeneRuler DNA Ladder Mix (ThermoFisher Scientific, cat. no. SM0331) was used in all the gels as a DNA size marker.

Figure 6.. Genotyping schematics.

(a) Genotyping floxed alleles. Primer sets 1–2 and 3–4 amplify single LoxP insertion at the two separate sites but cannot suggest if they are inserted in cis or in trans. Correct insertion genotype (in cis) can be determined by PCR using the primer sets 5–4 and 1–6, and confirmed by sequencing the PCR products. Note that the 3' ends of primers 5 & 6 bind to the first 15 bases of LoxP sites (primer 5;

5'-NNNNNNNNNNNNNNNNNNNNataacttcgtatagc-3'

: primer 6;

5'-NNNNNNNNNNNNNNNNNNNNataacttcgtataat-3'

). (b) Genotyping knock-in (and knock-down) alleles. Three PCRs are performed; one each for 5' and 3' junctional regions (primer sets 7–8 and 9–10), and the third PCR for insert-specific regions (primer set 12–11). PCR with outer primer sets (7–11 and 12–10) amplify longer PCR fragments, including the full knock-in cassette. If amplification of longer sequences is not successful (for example if primers 7–11 and 12–10 that amplify nearly the full length of the cassette do not work well), alternate primers within the insertion cassette should be tried. Similarly, alternate primers of 9 and 8 (within the cassette) can also be tried to obtain smaller amplicons (if larger products cannot be amplified efficiently for the junctional PCRs). The amplified fragments in both (a) and (b) should be sequenced to ensure sequence fidelity. The examples of PCRs of primer pairs 1–2 and 3–4 (for Pitx1 floxing), and 7–8 & 9–10 (MMP9-T2AmCitrine knock-in) were previously reported in Quadros et al., 2017[9]. All experimental procedures using mice was carried out according to Tokai University institutional regulations for animal usage (permit number: #165009).

Figure 7.. Assembling of MILLEX-GX 0.22 μM filter unit.

Insert a Millex-Gx 0.22 μM filter unit into a PCR tube (cut from an 8-well strip), insert the PCR tube containing the filter into a 1.5ml micro-centrifuge tube.

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