CLICK: one-step generation of conditional knockout mice

Microinjection of CRISPR/Cas9 with lssDNA to generate a floxed allele

The proof of concept experiment started with the design of two gRNAs targeting introns on either side of exon 4 of the serine peptidase inhibitor clade A member 3 N (Serpina3n) gene, and lssDNA, which contains exon 4 flanked by two loxP sequences with a 60-bp 3′ HA and an extended 300-bp 5′ HA to avoid the effect of possible 5′ exonuclease activity, as previously described [11] (Fig. 1a and Additional file 2: Figure S2). To increase Cas9 endonuclease activity, we also used Cas9-polyA plasmids (RIKEN BRC: RDB13130), which appended an 81-bp polyadenine tail to the transcription terminator [11]. Microinjection (MI) of two gRNAs (25 ng/μl each), Cas9-polyA mRNA (50 ng/l), and lssDNA (25 ng/μl) into 371 fertilized eggs from C57BL/6 mice resulted in 56 live births delivered from foster ICR mice (Table 1). PCR analysis of their tail tips using external primers outside HA sequences (Additional file 2: Figure S2) showed multiple bands, which appeared as mosaicism (more than three bands) in pups (Fig. 1b). Direct sequencing analysis of the PCR products demonstrated various insertions or deletions (indels) as well as large deletions (LDs) or inversions between the two cutting sites (Fig. 1c and Additional file 3: Figure S3). We found 16 pups carried loxP sequences, of which nine had two loxP sites floxing exon 4 (Table 1 and Fig. 1c). Among the 9 floxed mice (F0), 5 carried either homozygous floxed alleles or were heterozygous floxed with LD alleles, indicated as ‘Conditional’ knockouts in Table 1.

Species	Strain	Target gene	lssDNA size (bp)	Transfer method	Embryos injected (n)	Embryos transferred (%)	Live births (%)	F0 animals
								LD (%)	loxP (%)	flox (%)	Conditional (%)
Mouse	C57B6	Serpina3n	708	MI	371	255 (68.7)	56 (22.0)	28 (50.0)	7 (12.5)	9 (16.1)	5 (8.9)
	C57B6	Tyr	892	MI	222	149 (67.1)	17 (11.4)	2 (11.8)	4 (23.5)	3 (17.6)	–
	C57B6	mKIAA1322	1429	MI	166	134 (80.7)	5 (3.7)	1 (20.0)	–	4 (80.0)	2 (40.0)
	C57B6	Serpina3n	708	EL	180	160 (88.9)	18 (11.3)	9 (50.0)	2 (11.1)	2 (11.1)	2b (11.1)
	C57B6	Mct4	1095	EL	134	130 (97.0)	21 (16.2)	11 (52.4)	3 (14.3)	2 (9.5)	1 (4.7)
Rat	F344	Vapb	674	EL	160	77 (48.1)	6 (7.8)	2 (33.3)	1 (16.6)	3a (50.0)	1 (16.6)
Mouse	Emx1-cre	Serpina3n	708	MI	150	74 (49.3)	8 (10.8)	4 (50.0)	1 (12.5)	1 (12.5)	–
	Emx1-cre	Serpina3n	708	EL	150	113 (75.3)	27 (23.9)	10 (37.0)	1 (3.7)	5 (18.5)	3c (11.1)

Transfer method: gRNA/Cas9 and long single-stranded DNA (lssDNA) transferred by microinjection (MI) or electroporation (EL). Embryos transferred: two-cell embryos were transferred into a surrogate mother. LD: large fragments deleted between two gRNA targeting sites. loxP and flox: positive for either one or two loxP sites, respectively

^aAll three rats carried a missense mutation, P56S, together with floxed alleles. Conditional: conditional knockouts by homozygous floxed alleles or heterozygous floxed with LD alleles in F0 animals

^bSite-specific recombination confirmed by crossing with Cre-driver mice

^cSite-specific recombination confirmed in Cre-expressing tissues of F0 mice

- not identified

This method was repeated for exon 2 of the Tyrosinase (Tyr) gene and for exon 3 of the mKIAA1322 gene, which resulted in 3 mice carrying a floxed allele among 17 live births and 4 mice carrying a floxed allele among 5 live births, respectively (Table 1, Additional file 4: Figure S4, Additional file 5: Figure S5, Additional file 6: Figure S6, and Additional file 7: Figure S7). Crossing several F0 mice (#3, #22, #23 for floxed allele and #15, #20, #28 for single loxP-allele) with B6 mice confirmed the germline transmission of the floxed alleles into the next generation (Additional file 8: Figure 8). We observed no insertions or deletions at any off-target site for gRNAs targeting Serpina3n and Tyr genes across the whole genome in the F0 mice (Additional file 9: Figure 9).

Floxed alleles generated by zygote electroporation in mice and rats

We have previously reported the easy and efficient generation of knockout (KO) or knock-in (KI) mice using the CRISPR/Cas9 system with zygote electroporation (EL) instead of MI [12, 13]. For KI of floxed alleles, we used lssDNA with CRISPR/Cas9 for the transfer into embryos by electroporation. The zygote EL of two gRNAs (100 ng/μl each), Cas9 mRNA (400 ng/μl), and lssDNA (40 ng/μl) for Serpina3n or Monocarboxylate transporter 4 (Mct4) genes into 180 and 134 fertilized eggs resulted in 18 and 21 live births, respectively (Table 1). PCR and sequence analysis demonstrated carriers with various mutations, indels, LDs, and floxed alleles (Additional file 10: Figure S10, Additional file 11: Figure S11 and Additional file 12: Figure S12). The live birth rate, indel mutation rate, and loxP insertion efficiency were similar to those of MI, except for the higher survival rates of two-cell embryos (80–90%) by EL, as previously reported [12, 13] (Table 1). Moreover, crossing the floxed mice with Cre-driver mice (B6.Cg-Tg(CAG-Cre)CZ-MO2Osb: RBRC01828) provided site-specific recombination events between the two loxP sites, which confirmed that the floxed alleles were functional (Additional file 13: Figure S13). Therefore, we named this method CLICK: CRISPR with lssDNA inducing conditional knockout alleles (Fig. 2a).

To test whether CLICK can be applied to other species such as rats, we designed lssDNA including two loxP sequences floxing exon 2 of the rat vesicle-associated membrane protein-associated protein B/C (Vapb) gene with a P56S mutation, which is associated with amyotrophic lateral sclerosis in humans [14] (Fig. 2b and Additional file 14: Figure S14). EL of the lssDNA and CRISPR components into rat zygotes resulted in the birth of six pups, which contained indels, LD, and inversion mutations between the cut sites (Table 1, Fig. 2c and d). Rats #1, #2, and #6 showed floxed alleles with the P56S mutation. Crossing these F0 rats with F344 rats or Cre-driver rats (W-Tg(CAG-cre)81Jmsk (NBRP-Rat No.0283)) provided the transmission of the floxed alleles or site-specific recombination that removed exon 2, respectively (Additional file 8: Figure S8 and Additional file 13: Figure S13).

One-step generation of conditional knockout animals in the founder generation

Finally, we applied the CLICK method to in vitro fertilized eggs of B6 mice with the sperm of Cre-driver mice (Emx1-cre: RBRC00808) expressing Cre in the cortical neurons and glia [15] (Fig. 3a). Among 8 and 27 live births by MI and EL, respectively, 6 mice carried floxed alleles for exon 2 of the Serpina3n gene (Table 1, Fig. 3b and Additional file 15: Table S1). Interestingly, 3 floxed mice, #14, #16, and #24, carried homozygous floxed alleles or heterozygous floxed with LD alleles in addition to the Emx1-Cre allele. PCR analysis of multiple tissues of #14 indicated a brain-specific recombination that removed exon 2 of the Serpina3n gene in this animal (Fig. 3c). These results indicate that CLICK can be used for the one-step generation of conditional knockout mice (F0) without further crossing, using fertilized eggs expressing tissue-specific Cre (Fig. 3d and Additional file 16: Table S2).

Animals

C57BL/6JJcl mice and F344/Jcl rats were obtained from CLEA Japan Inc., Tokyo, Japan. B6.Cg-Tg(CAG-Cre)CZ-MO2Osb (RBRC01828) and Emx1-cre (RBRC00808) mice were provided by RIKEN BRC (www.en.brc.riken.jp), and W-Tg(CAG-cre)81Jmsk (NBRP-Rat No.0283) rats were from the National Bio Resource Project for the Rat in Japan (www.anim.med.kyoto-u.ac.jp/nbr). The animals were kept under conditions of 50% humidity and a 12:12 h light:dark cycle. They were fed a standard pellet diet (MF, Oriental Yeast Co., Tokyo, Japan) and tap water ad libitum.

Preparation of Cas9, gRNA and lssDNAs

We formally constructed pCas9-polyA and deposited it into the Addgene repository (ID #72602; www.addgene.org/CRISPR). mRNA was transcribed in vitro using a mMESSAGE mMACHINE T7 Ultra Kit (Life Technologies, Carlsbad, CA, USA) from linearized plasmids and was purified using a MEGAClear kit (Life Technologies). To design gRNAs, software tools (www.crispr.genome-engineering.org) predicting unique target sites throughout the mouse and rat genome were used. gRNAs were transcribed in vitro using a MEGAshortscript T7 Transcription Kit (Life Technologies) from synthetic double-strand DNAs obtained from IDT (Integrated DNA Technologies, IA, USA) or Life Technologies.

lssDNAs were prepared by a simple method using nicking endonucleases as we previously reported [11] (Additional file 1: Figure S1). Briefly, double-stranded DNA plasmids comprising of a floxed allele, homology arms, and two nicking endonuclease sites were obtained from Thermo Fisher Scientific (MA, USA) as GeneArt^® Gene Synthesis. For digestion, 100 μg of the purified plasmid DNA was incubated at an optimum temperature for 2 to 3 h with nicking endonucleases, such as Nt.BspQI and Nb.BbvCI (New England Biolabs Inc., MA, USA). After purification by ethanol precipitation, the DNAs were denatured with 3-fold amounts of formamide (Nacalai Tesque, Inc., Tokyo, Japan) at 80 °C for 10 min, and then subjected to agarose gel electrophoresis with DynaMarker^®Prestain Marker for RNA High concentrations (Biodynamics Laboratory Inc., Tokyo, Japan). Bands corresponding to a single-strand DNA fragment were extracted using NucleoSpin^® Gel and PCR Clean-up (Takara Bio, Shiga, Japan). Finally, 2–4 μg of lssDNA was obtained with this method.

Microinjection and electroporation into mouse and rat embryos

Pronuclear-stage mouse embryos were prepared by thawing frozen embryos (CLEA Japan Inc.), or in vitro fertilized embryos, or fresh embryos collected from naturally mated female mice that were superovulated by injection with pregnant mare serum gonadotropin (PMSG: ASKA Animal Health Co., Tokyo, Japan) and human chorionic gonadotropin (HCG: ASKA Animal Health Co.). Rat embryos were collected from 8 to 12 weeks of age females that were superovulated by the administration of 150 U/kg of PMSG followed by 75 U/kg of HCG. After natural mating, pronuclear-stage embryos were collected from the oviducts of the females and cultured in a modified Krebs–Ringer bicarbonate medium or KSOM medium (ARK Resource, Kumamoto, Japan).

In MI, 50 ng/μl Cas9 mRNA, 25 ng/μl gRNA, or 50 ng/μl lssDNA were microinjected into the male pronuclei of embryos using a micromanipulator (Narishige, Tokyo, Japan). In targeting mouse KIAA1322, 30 ng/μl Cas9 protein (PNA Bio, Thousand Oaks, CA, USA) was used instead of Cas9 mRNA [21]. This was cultured in modified Krebs–Ringer bicarbonate or KSOM medium overnight and divided two-cell embryos were transferred into pseudopregnant females.

For EL, 50–100 embryos at 1 h after thawing or 3–4 h after collection were placed into a chamber with 40 μl of serum free media (Opti-MEM, Thermo Fisher Scientific) containing 400 ng/μl Cas9 mRNA, 200 ng/μl gRNA, or 40 ng/μl lssDNA. They were electroporated with a 5 mm gap electrode (CUY505P5 or CUY520P5 Nepa Gene, Chiba, Japan) in a NEPA21 Super Electroporator (Nepa Gene, Chiba, Japan).The poring pulses for the electroporation were voltage 225 V, pulse width 1.5 ms for mouse embryos and 2.0 ms for rat embryos, pulse interval 50 ms, and number of pulses 4. The first and second transfer pulses were voltage 20 V, pulse width 50 ms, pulse interval 50 ms, and number of pulses 5. Mouse or rat embryos that developed to the two-cell stage after the introduction of RNA and lssDNA were transferred into the oviducts of female surrogates anesthetized with isoflurane (DS Pharma Animal Health Co., Ltd., Osaka, Japan).

Genotyping analysis

Genomic DNA was extracted from the tail tip using the KAPA Express Extract DNA Extraction Kit (Kapa Biosystems, London, UK). For PCR and sequence analysis, we used external primers outside the HA, which amplied the targeted region (Additional file 18: Table S4). PCR was performed in a total volume of 15 μl under the following conditions: 1 cycle of 94 °C for 3 min; 35 cycles of 94 °C for 30 s, 60 °C for 1 min and 72 °C for 45 s; and 1 cycle of 72 °C for 3 min. The final reaction mixture contained 200 μM dNTPs, 1.0 mM MgCl₂, and 0.66 μM of primer. The PCR products were then directly sequenced using the BigDye Terminator v3.1 cycle sequencing mix and the standard protocol for an Applied Biosystems 3130 DNA Sequencer (Life Technologies). To confirm mosaic mutations, we sequenced individual TA clones in some cases.