BACKGROUND
gene-modified  mice are essential resources for studying gene function, developmental processes, and human diseases  <cit> .
the production of gm mice, such as conditional knockout  mice, requires several time-consuming steps.
for example, a cko vector carrying the gene of interest and a drug-resistant gene must be constructed.
the constructed cko vector is then introduced into the genome of embryonic stem  cells via homologous recombination.
the resulting cko cells are used to produce a chimeric mouse, with which germ-line transmission is achieved via es-derived germ cells.
finally, offspring with the ko phenotype are obtained after genotyping and breeding.
in early  <dig>  a novel genome editing method, called the “clustered regulatory interspaced short palindromic repeat /crispr-associated protein  <dig>  system”, was reported.
the crispr/cas <dig> system enables the efficient production of biallelic  ko cells/embryos  <cit> .
the system comprises crispr-coded rnas , trans-activating crrnas , and cas <dig> endonuclease.
it was originally identified as an adaptive immune apparatus in bacteria and archaea for defense against invading foreign plasmids and phages  <cit> .
a working unit of the crispr/cas <dig> complex  and humanized cas <dig> protein) was established to enable the system to work effectively in mammalian cells  <cit> .
the system efficiently introduces double-strand breaks approximately 3 bp in front of a protospacer adjacent motif  sequence , which is downstream of the grna target sequence  + pam)  <cit> .
when the target sequence is repaired by non-homologous end joining , an insertion and deletion  mutation results.
when a construct with homology to the sequences upstream and downstream of the grna target sequence, such as a cko vector, is introduced with the cas <dig> protein, homologous recombination  occurs  <cit> .
the most attractive aspect of the crispr/cas <dig> system is the efficient production of biallelic ko offspring through the direct injection of mrna encoding cas <dig> and grna.
wang et al.
<cit>  demonstrated that 57–100% of injected zygotes were biallelic ko, when fetuses derived from the microinjected zygotes were examined.
several researchers have confirmed these results and shown that a high rate of indel mutations can be achieved using the crisp/cas <dig> system in mice  <cit> .
however, a major problem associated with this system is the need to confirm the presence of the indel mutation in the target sequence in order to know whether the samples possess the ko genotype.
to this end, dna from fetuses or newborn mice is frequently assessed by nuclease assays and sequencing analysis  <cit> .
for optimization of the crsipr/cas9-based gm mouse system, examining blastocysts at the pre-implantation stage is desirable because the evaluation can be performed 3 days after microinjection and does not require egg transfer to recipients to produce offspring.
unfortunately, few studies have assessed indel mutations at this stage , probably because extremely low amounts of dna are recovered at this stage and the collected dna is occasionally lost.
in this study, we used a single mouse blastocyst as a genomic dna source for several mutational assays.
to establish a sensitive and reproducible assay for the detection of mutations introduced by crispr/cas9-mediated genome editing, we first checked whether t <dig> endonuclease i could be used as an alternative to the widely utilized surveyor nuclease.
we next microinjected crispr/cas9-related rna/dna into fertilized eggs and prepared an optimized crude dna solution from developed blastocysts to examine nhej-mediated knockout and hr-mediated knockin of the endogenous receptor  activity modifying protein  <dig>  gene.
in addition, we used whole genome amplification  technology  <cit> , capable of producing microgram quantities of genomic dna from a very small amount of dna, to see whether dna amplified from the crude dna extract of a single blastocyst provided an appropriate template for polymerase chain reaction  analysis.
our results will be useful for researchers and technicians who want to analyze indel mutations at the blastocyst stage.
RESULTS
t <dig> endonuclease i is more effective than surveyor nuclease for detecting indel mutations
to evaluate which nuclease, surveyor nuclease or t <dig> endonuclease i, provided clearer and more reproducible results, p <dig> and p <dig> pcr products  were used as templates .
the p <dig> and p <dig> products were derived from pcr of pc2epa and pcepa, respectively, which are nearly identical .
a mixture containing p <dig>  and p <dig>  , p <dig>  and p <dig>   or p <dig>  and p <dig>   was re-annealed and treated with each nuclease .
a mixture containing p <dig>  and p <dig>  that had been directly treated with nuclease without re-annealing was used as a control .
when the pcr products were subjected to electrophoresis,  <dig> expected cleavage bands of  <dig> and 275 bp, generated by nuclease treatment, were observed .
notably, the bands produced by t <dig> endonuclease i were more distinct than those generated by surveyor nuclease.
the weak cleavage activity of surveyor nuclease improved slightly when the amount of surveyor nuclease added to the reaction mixture was increased or the reaction time was extended .
furthermore, t <dig> endonuclease i produced clearer bands than did surveyor nuclease when the nuclease assay was performed using pcr-amplified products derived from a single blastocyst .
these results suggest that t <dig> endonuclease i is superior to surveyor nuclease for detecting indel mutations in target dna.
in the following experiments, we used t <dig> endonuclease i to detect indel mutations.
optimization of crude dna template preparation from a single blastocyst for highly reproducible direct pcr amplification
to achieve highly reproducible pcr amplification using a single blastocyst as the source of the crude dna template, we first checked several carriers, including yeast trna , yeast total rna , herring sperm dna , and glycogen , all of which have been used to precipitate very low amounts of dna.
in preliminary tests, yeast trna was better than the other reagents, when evaluated in view of successful pcr amplification .
therefore, we used yeast trna to prepare a crude dna solution  from a single blastocyst for use in the nuclease assay, as described in the methods section.
we successfully amplified the murine ramp <dig> gene from total  <dig> individual blastocysts ; see  <cit>  for information on the function of ramp2).
1
2
1injection was performed toward cyto: cytoplasm; pn/cyto pronuclei and cytoplasm.
2mutated blastocysts were identified by both t7endonucleasei digestion and sequencing.
3representative results from experiment  <dig> are shown in figure 2b and c.
1
1injection was performed toward pn: pronuclei; cyto: cytoplasm; pn/cyto: pronuclei and cytopalsm.
2representative results from experiment  <dig> are shown in figure 4b and c.
use of the single blastocyst-based assay to detect indel mutations introduced by crispr/cas9-mediated genome editing in endogenous murine ramp2
as shown in figure  <dig>  we developed a single blastocyst-based mutational assay using t <dig> endonuclease i and a crude dna solution.
next, we tested whether the mutational assay could detect mutations in exon  <dig> of endogenous murine ramp <dig> after microinjection of cas <dig> mrna and ramp <dig> grna   at the zygote stage.
representative results are shown in figure 2b and c and in table  <dig> .
first, approximately  <dig> pl of an rna mixture  was injected into the pronuclei and cytoplasm of  <dig> fertilized mouse eggs .
the  <dig> eggs that survived after injection were cultivated to the blastocyst stage.
crude dna was then prepared by lysing each of the  <dig> blastocysts derived from the rna-injected zygotes  and  <dig> blastocyst derived from a non-injected fertilized egg as a negative control .
finally, pcr was performed using each crude dna sample as template to specifically amplify a 580-bp region corresponding to exon  <dig> of murine ramp <dig> .
pcr products were successfully amplified from all of the samples tested .
two of the samples produced  <dig> major bands: unexpected and expected around 580 bp , and  <dig> of the samples yielded a pcr product slightly shorter than that of the others .
since ramp <dig> has two alleles, these results indicated that the samples in lanes  <dig> and  <dig> had relatively long deletions in a heterozygous state or a mosaic state, while the sample in lane  <dig> had a short deletion in a homozygous state.
to obtain more information on the indel mutations in the  <dig> samples , each pcr product was digested with t <dig> endonuclease i as described in the methods section, and the resulting fragments were sequenced .
as expected, the pcr products  derived from nic sample were undigestable.
sequencing of these products revealed no mutations  sequence in figure 2c).
pcr products derived from  <dig> blastocysts  produced an additional  <dig> to  <dig> bands.
sequencing of the pcr product shown in lane  <dig> revealed a 55-bp heterozygous deletion, located at − <dig> to − <dig>  where the third nucleotide  of the pam motif  is considered position  <dig> .
similarly, the pcr products in lanes  <dig>   <dig>   <dig>  and  <dig> contained a 206-bp heterozygous deletion , 10-bp heterozygous deletion , 4-bp heterozygous deletion , and 3-bp heterozygous deletion , respectively.
for pcr products in lane  <dig>  both 23-bp deletion  of two alleles and 372-bp deletion  of one allele were revealed.
the pcr products in lanes  <dig> and  <dig> were not digested by t <dig> endonuclease i; only  <dig> major band was apparent after nuclease digestion.
however, sequencing demonstrated that the samples in lanes  <dig> and  <dig> had a 6-bp homozygous deletion  and 43-bp homozygous deletion , respectively.
thus, all of the  <dig> blastocysts tested had indel mutations in the murine ramp <dig> gene .
heterozygous deletions occurred in  <dig> blastocysts .
homozygous deletions occurred in  <dig> blastocysts .
these results showed that the t <dig> endonuclease i-based assay using crude dna prepared from a single blastocyst is a sensitive and reproducible method for the detection of indel mutations in a target gene.
in addition, we tested whether the single blastocyst-based assay could detect an hr event in exon  <dig> of murine ramp <dig>  induced by the crispr/cas <dig> system .
first, approximately  <dig> pl of an rna mixture  was injected into the pronuclei and cytoplasm of  <dig> fertilized mouse eggs.
of these,  <dig> live eggs were cultivated to the blastocyst stage.
seven embryos developed successfully to blastocysts, of which  <dig> were egfp-positive.
in figure 4b,  <dig> of the  <dig> fluorescent blastocysts are shown.
next, we prepared crude dna solutions by lysing each of the  <dig> blastocysts .
the presence or absence of hr in the target locus was assessed by pcr amplification of the junctional region between the 5′ arm  and the host genome  using crude dna as template.
as shown in figure 4c, each of the  <dig> egfp-positive blastocysts had pcr products with an expected size of approximately  <dig>  kb, but the other samples, including the  <dig> egfp-negative blastocysts and the  <dig> uninjected blastocyst, did not.
thus, hr occurred successfully in the  <dig> egfp-positive samples, and the hr rate was  <dig> % .
these results indicate that single blastocysts are useful for the detection hr events induced by crispr/cas9-mediated genome editing.
crude dna from a single blastocyst is an appropriate template for wga and subsequent pcr analysis
the main problem when using the t <dig> endonuclease i-based assay with a single blastocyst is that the dna content is very small, which often hampers extensive analysis of the target dna region.
for example, we typically prepare a 10-μl crude dna solution from  <dig> blastocyst, which is enough for  <dig> pcr reactions .
although this amount of crude dna is presently sufficient for analyzing on-target mutations, it is not adequate for examining additional areas to identify possible off-target mutation sites.
to resolve this problem, we used wga, a method for the robust amplification of an entire genome starting with nanogram quantities of dna  <cit> .
when crude dna solutions prepared from each of the  <dig> blastocysts  were subjected to wga, 3–4 μg of genomic dna was successfully amplified .
when pcr was performed using the dna generated from wga as template, a 580-bp product corresponding to exon  <dig> of murine ramp <dig> was successfully amplified .
notably, the electrophoretic pattern of the pcr products was similar to that of the pcr products obtained from direct amplification of the crude dna solutions .
when the pcr products derived from wga of the crude dna were re-annealed and subsequently digested with t <dig> endonuclease i, the digestion pattern of the pcr products was similar to that of pcr products derived from direct amplification of the crude dna solution .
in the negative control, in which water was subjected to wga , a small amount of dna was observed, but pcr using the ntc dna as template did not generate detectable pcr products , as previously suggested by akasaka et al.
<cit> .
these results indicate that a crude dna solution prepared from a single blastocyst can serve as template for wga.
amplification of genomic dna by wga may be an effective approach when extensive analyses of on- or off-target mutations are required.
however, since wga has a potential risk of contaminations and the introduction of unwanted mutations  <cit> , wga may require careful evaluation of the results.
direct microinjection of crispr/cas9-related nucleic acids into zygotes is a powerful tool for the efficient production of gm animals, including mice  <cit> , rats  <cit> , and monkeys  <cit> .
in most cases, newborn mice or fetuses have been used to confirm the presence of indel mutations in the genome  <cit> .
however, this requires egg transfer to recipients, which is a time-consuming process.
the single blastocyst-based assay described here will allow rapid determination of whether the crispr/cas <dig> system designed by the practitioners is able to generate gm mice, as indicated in tables  <dig> and  <dig>  furthermore, the assay does not require preliminary tests to check the effectiveness of crispr/cas9-related nucleic acids via the transfection of cultured cells.
moreover, if practitioners transfer microinjected zygotes to recipients and do not obtain gm mice, our assay provides a way to determine the reason, for example, fetal lethality caused by a crispr/cas9-mediated mutation or low efficiency of the crispr/cas9-related rna/dna.
the present study demonstrated that the crude dna extracted from a single blastocyst is sufficient for the detection of mutations in nuclease assays that used t <dig> endonuclease i  as an alternative to the widely utilized surveyor nuclease  <cit> .
a few researchers have reported the usefulness of t <dig> endonuclease i for detecting mutations in a target gene  <cit> .
using the single blastocyst assay with t <dig> endonuclease i and direct sequencing analysis, we demonstrated that microinjection of crispr/cas9-related nucleic acids into zygotes leads to a relatively high degree of mutation , as previously described by wang et al.
<cit> .
we also demonstrated that the crude dna sample is sufficient for the detection of hr in the ramp <dig> locus , as previously demonstrated by yang et al.
<cit> .
notably, using the present assay and even one experiment with a small number  of zygotes, researchers and technicians can assess the mutation rate and post-injection embryo survival.
this assay also will be helpful to support the animal welfare since the number of mice used in research can be reduced.
furthermore, we demonstrated the usefulness of wga before pcr and the subsequent nuclease assay .
wga amplifies nanogram levels of genomic dna from a single blastocyst to microgram levels, which enables multiple analyses of genomic dna, as previously noted by akasaka et al.
<cit> .
the single blastocyst-based assay described in this study is applicable to the evaluation of mutations generated by the other gnome editing systems, such as zinc-finger nuclease   <cit>  and transcription activator-like nuclease   <cit> , because dna modification through nhej and hr with these systems is principally the same as with the crispr/cas <dig> system.
CONCLUSIONS
we have demonstrated the usefulness of a single blastocyst-based assay for detecting mutations introduced by the crispr/cas <dig> system.
this assay, which is reproducible and convenient, allows researchers and technicians to check the effectiveness of crspr/cas9-based genome editing rapidly.

 gene-modified mice are essential resources for studying gene function, developmental processes, and human diseases. the production of gm mice, such as conditional knockout mice, requires several time-consuming steps. for example, a cko vector carrying the gene of interest and a drug-resistant gene must be constructed. the constructed cko vector is then introduced into the genome of embryonic stem cells via homologous recombination. the resulting cko cells are used to produce a chimeric mouse, with which germ-line transmission is achieved via es-derived germ cells. finally, offspring with the ko phenotype are obtained after genotyping and breeding. in early a novel genome editing method, called the “clustered regulatory interspaced short palindromic repeat /crispr-associated protein system”, was reported. the crispr/cas system enables the efficient production of biallelic ko cells/embryos. the system comprises crispr-coded rnas, trans-activating crrnas, and cas endonuclease. it was originally identified as an adaptive immune apparatus in bacteria and archaea for defense against invading foreign plasmids and phages. a working unit of the crispr/cas complex and humanized cas protein) was established to enable the system to work effectively in mammalian cells. the system efficiently introduces double-strand breaks approximately 3 bp in front of a protospacer adjacent motif sequence, which is downstream of the grna target sequence  + pam). when the target sequence is repaired by non-homologous end joining, an insertion and deletion mutation results. when a construct with homology to the sequences upstream and downstream of the grna target sequence, such as a cko vector, is introduced with the cas protein, homologous recombination occurs. the most attractive aspect of the crispr/cas system is the efficient production of biallelic ko offspring through the direct injection of mrna encoding cas and grna. wang et al. demonstrated that 57–100% of injected zygotes were biallelic ko, when fetuses derived from the microinjected zygotes were examined. several researchers have confirmed these results and shown that a high rate of indel mutations can be achieved using the crisp/cas system in mice. however, a major problem associated with this system is the need to confirm the presence of the indel mutation in the target sequence in order to know whether the samples possess the ko genotype. to this end, dna from fetuses or newborn mice is frequently assessed by nuclease assays and sequencing analysis. for optimization of the crsipr/cas9-based gm mouse system, examining blastocysts at the pre-implantation stage is desirable because the evaluation can be performed 3 days after microinjection and does not require egg transfer to recipients to produce offspring. unfortunately, few studies have assessed indel mutations at this stage, probably because extremely low amounts of dna are recovered at this stage and the collected dna is occasionally lost. in this study, we used a single mouse blastocyst as a genomic dna source for several mutational assays. to establish a sensitive and reproducible assay for the detection of mutations introduced by crispr/cas9-mediated genome editing, we first checked whether t endonuclease i could be used as an alternative to the widely utilized surveyor nuclease. we next microinjected crispr/cas9-related rna/dna into fertilized eggs and prepared an optimized crude dna solution from developed blastocysts to examine nhej-mediated knockout and hr-mediated knockin of the endogenous receptor activity modifying protein gene. in addition, we used whole genome amplification technology, capable of producing microgram quantities of genomic dna from a very small amount of dna, to see whether dna amplified from the crude dna extract of a single blastocyst provided an appropriate template for polymerase chain reaction analysis. our results will be useful for researchers and technicians who want to analyze indel mutations at the blastocyst stage.  t endonuclease i is more effective than surveyor nuclease for detecting indel mutations to evaluate which nuclease, surveyor nuclease or t endonuclease i, provided clearer and more reproducible results, p and p pcr products were used as templates. the p and p products were derived from pcr of pc2epa and pcepa, respectively, which are nearly identical. a mixture containing p and p, p and p or p and p was re-annealed and treated with each nuclease. a mixture containing p and p that had been directly treated with nuclease without re-annealing was used as a control. when the pcr products were subjected to electrophoresis, expected cleavage bands of and 275 bp, generated by nuclease treatment, were observed. notably, the bands produced by t endonuclease i were more distinct than those generated by surveyor nuclease. the weak cleavage activity of surveyor nuclease improved slightly when the amount of surveyor nuclease added to the reaction mixture was increased or the reaction time was extended. furthermore, t endonuclease i produced clearer bands than did surveyor nuclease when the nuclease assay was performed using pcr-amplified products derived from a single blastocyst. these results suggest that t endonuclease i is superior to surveyor nuclease for detecting indel mutations in target dna. in the following experiments, we used t endonuclease i to detect indel mutations. optimization of crude dna template preparation from a single blastocyst for highly reproducible direct pcr amplification to achieve highly reproducible pcr amplification using a single blastocyst as the source of the crude dna template, we first checked several carriers, including yeast trna, yeast total rna, herring sperm dna, and glycogen, all of which have been used to precipitate very low amounts of dna. in preliminary tests, yeast trna was better than the other reagents, when evaluated in view of successful pcr amplification. therefore, we used yeast trna to prepare a crude dna solution from a single blastocyst for use in the nuclease assay, as described in the methods section. we successfully amplified the murine ramp gene from total individual blastocysts; see for information on the function of ramp2). 1 2 1injection was performed toward cyto: cytoplasm; pn/cyto pronuclei and cytoplasm. 2mutated blastocysts were identified by both t7endonucleasei digestion and sequencing. 3representative results from experiment are shown in figure 2b and c. 1 1injection was performed toward pn: pronuclei; cyto: cytoplasm; pn/cyto: pronuclei and cytopalsm. 2representative results from experiment are shown in figure 4b and c. use of the single blastocyst-based assay to detect indel mutations introduced by crispr/cas9-mediated genome editing in endogenous murine ramp2 as shown in figure  we developed a single blastocyst-based mutational assay using t endonuclease i and a crude dna solution. next, we tested whether the mutational assay could detect mutations in exon of endogenous murine ramp after microinjection of cas mrna and ramp grna at the zygote stage. representative results are shown in figure 2b and c and in table . first, approximately pl of an rna mixture was injected into the pronuclei and cytoplasm of fertilized mouse eggs. the eggs that survived after injection were cultivated to the blastocyst stage. crude dna was then prepared by lysing each of the blastocysts derived from the rna-injected zygotes and blastocyst derived from a non-injected fertilized egg as a negative control. finally, pcr was performed using each crude dna sample as template to specifically amplify a 580-bp region corresponding to exon of murine ramp. pcr products were successfully amplified from all of the samples tested. two of the samples produced major bands: unexpected and expected around 580 bp, and of the samples yielded a pcr product slightly shorter than that of the others. since ramp has two alleles, these results indicated that the samples in lanes and had relatively long deletions in a heterozygous state or a mosaic state, while the sample in lane had a short deletion in a homozygous state. to obtain more information on the indel mutations in the samples, each pcr product was digested with t endonuclease i as described in the methods section, and the resulting fragments were sequenced. as expected, the pcr products derived from nic sample were undigestable. sequencing of these products revealed no mutations sequence in figure 2c). pcr products derived from blastocysts produced an additional to bands. sequencing of the pcr product shown in lane revealed a 55-bp heterozygous deletion, located at − to − where the third nucleotide of the pam motif is considered position. similarly, the pcr products in lanes and contained a 206-bp heterozygous deletion, 10-bp heterozygous deletion, 4-bp heterozygous deletion, and 3-bp heterozygous deletion, respectively. for pcr products in lane both 23-bp deletion of two alleles and 372-bp deletion of one allele were revealed. the pcr products in lanes and were not digested by t endonuclease i; only major band was apparent after nuclease digestion. however, sequencing demonstrated that the samples in lanes and had a 6-bp homozygous deletion and 43-bp homozygous deletion, respectively. thus, all of the blastocysts tested had indel mutations in the murine ramp gene. heterozygous deletions occurred in blastocysts. homozygous deletions occurred in blastocysts. these results showed that the t endonuclease i-based assay using crude dna prepared from a single blastocyst is a sensitive and reproducible method for the detection of indel mutations in a target gene. in addition, we tested whether the single blastocyst-based assay could detect an hr event in exon of murine ramp induced by the crispr/cas system. first, approximately pl of an rna mixture was injected into the pronuclei and cytoplasm of fertilized mouse eggs. of these, live eggs were cultivated to the blastocyst stage. seven embryos developed successfully to blastocysts, of which were egfp-positive. in figure 4b, of the fluorescent blastocysts are shown. next, we prepared crude dna solutions by lysing each of the blastocysts. the presence or absence of hr in the target locus was assessed by pcr amplification of the junctional region between the 5′ arm and the host genome using crude dna as template. as shown in figure 4c, each of the egfp-positive blastocysts had pcr products with an expected size of approximately  kb, but the other samples, including the egfp-negative blastocysts and the uninjected blastocyst, did not. thus, hr occurred successfully in the egfp-positive samples, and the hr rate was %. these results indicate that single blastocysts are useful for the detection hr events induced by crispr/cas9-mediated genome editing. crude dna from a single blastocyst is an appropriate template for wga and subsequent pcr analysis the main problem when using the t endonuclease i-based assay with a single blastocyst is that the dna content is very small, which often hampers extensive analysis of the target dna region. for example, we typically prepare a 10-μl crude dna solution from blastocyst, which is enough for pcr reactions. although this amount of crude dna is presently sufficient for analyzing on-target mutations, it is not adequate for examining additional areas to identify possible off-target mutation sites. to resolve this problem, we used wga, a method for the robust amplification of an entire genome starting with nanogram quantities of dna. when crude dna solutions prepared from each of the blastocysts were subjected to wga, 3–4 μg of genomic dna was successfully amplified. when pcr was performed using the dna generated from wga as template, a 580-bp product corresponding to exon of murine ramp was successfully amplified. notably, the electrophoretic pattern of the pcr products was similar to that of the pcr products obtained from direct amplification of the crude dna solutions. when the pcr products derived from wga of the crude dna were re-annealed and subsequently digested with t endonuclease i, the digestion pattern of the pcr products was similar to that of pcr products derived from direct amplification of the crude dna solution. in the negative control, in which water was subjected to wga, a small amount of dna was observed, but pcr using the ntc dna as template did not generate detectable pcr products, as previously suggested by akasaka et al.. these results indicate that a crude dna solution prepared from a single blastocyst can serve as template for wga. amplification of genomic dna by wga may be an effective approach when extensive analyses of on- or off-target mutations are required. however, since wga has a potential risk of contaminations and the introduction of unwanted mutations, wga may require careful evaluation of the results. direct microinjection of crispr/cas9-related nucleic acids into zygotes is a powerful tool for the efficient production of gm animals, including mice, rats, and monkeys. in most cases, newborn mice or fetuses have been used to confirm the presence of indel mutations in the genome. however, this requires egg transfer to recipients, which is a time-consuming process. the single blastocyst-based assay described here will allow rapid determination of whether the crispr/cas system designed by the practitioners is able to generate gm mice, as indicated in tables  and furthermore, the assay does not require preliminary tests to check the effectiveness of crispr/cas9-related nucleic acids via the transfection of cultured cells. moreover, if practitioners transfer microinjected zygotes to recipients and do not obtain gm mice, our assay provides a way to determine the reason, for example, fetal lethality caused by a crispr/cas9-mediated mutation or low efficiency of the crispr/cas9-related rna/dna. the present study demonstrated that the crude dna extracted from a single blastocyst is sufficient for the detection of mutations in nuclease assays that used t endonuclease i as an alternative to the widely utilized surveyor nuclease. a few researchers have reported the usefulness of t endonuclease i for detecting mutations in a target gene. using the single blastocyst assay with t endonuclease i and direct sequencing analysis, we demonstrated that microinjection of crispr/cas9-related nucleic acids into zygotes leads to a relatively high degree of mutation, as previously described by wang et al.. we also demonstrated that the crude dna sample is sufficient for the detection of hr in the ramp locus, as previously demonstrated by yang et al.. notably, using the present assay and even one experiment with a small number of zygotes, researchers and technicians can assess the mutation rate and post-injection embryo survival. this assay also will be helpful to support the animal welfare since the number of mice used in research can be reduced. furthermore, we demonstrated the usefulness of wga before pcr and the subsequent nuclease assay. wga amplifies nanogram levels of genomic dna from a single blastocyst to microgram levels, which enables multiple analyses of genomic dna, as previously noted by akasaka et al.. the single blastocyst-based assay described in this study is applicable to the evaluation of mutations generated by the other gnome editing systems, such as zinc-finger nuclease and transcription activator-like nuclease, because dna modification through nhej and hr with these systems is principally the same as with the crispr/cas system.  we have demonstrated the usefulness of a single blastocyst-based assay for detecting mutations introduced by the crispr/cas system. this assay, which is reproducible and convenient, allows researchers and technicians to check the effectiveness of crspr/cas9-based genome editing rapidly. 
individual blastocysts were lysed to obtain crude dna solutions, which were used for polymerase chain reaction assays. mouse fertilized eggs were microinjected with crispr/cas9-related rna/dna to examine whether non-homologous end joining-mediated knockout and homologous recombination-mediated knockin occurred in the endogenous receptor activity modifying protein gene. t endonuclease i was more effective than surveyor nuclease for detecting mutations in annealed fragments derived from plasmids, which contained nearly identical sequences. our results also established that crude dna from a single blastocyst was an appropriate template for whole genome amplification and subsequent assessment by pcr and the t endonuclease i-based assay. crispr/cas9gene targetingmicroinjectionblastocystt endonuclease isurveyor assaycrude dna solutionwhole genome amplification microinjection of clustered regulatory interspaced short palindromic repeat /crispr-associated protein -related rna and dna into fertilized eggs is a novel approach for creating gene-modified mice. however, it is not known whether indel mutations caused by the crispr/cas system can be reproducibly detected in embryos.
 microinjection of clustered regulatory interspaced short palindromic repeat /crispr-associated protein -related rna and dna into fertilized eggs is a novel approach for creating gene-modified mice. blastocysts obtained just before implantation may be appropriate for testing the fidelity of cripsr/cas9-mediated genome editing because they can be individually handled in vitro and obtained 3 days after microinjection, thus allowing researchers to check mutations rapidly. however, it is not known whether indel mutations caused by the crispr/cas system can be reproducibly detected in embryos. in this study, we assessed the detection of crispr/cas9-induced mutations in embryos.  t endonuclease i was more effective than surveyor nuclease for detecting mutations in annealed fragments derived from plasmids, which contained nearly identical sequences. mouse fertilized eggs were microinjected with crispr/cas9-related rna/dna to examine whether non-homologous end joining-mediated knockout and homologous recombination-mediated knockin occurred in the endogenous receptor activity modifying protein gene. individual blastocysts were lysed to obtain crude dna solutions, which were used for polymerase chain reaction assays. t endonuclease i-based pcr and sequencing analysis demonstrated that 25–100% of the embryos were knockout embryos and 7–57% of the embryos were knockin embryos. our results also established that crude dna from a single blastocyst was an appropriate template for whole genome amplification and subsequent assessment by pcr and the t endonuclease i-based assay.  the single blastocyst-based assay was useful for determining whether crispr/cas9-mediated genome editing worked in murine embryos. crispr/cas9gene targetingmicroinjectionblastocystt endonuclease isurveyor assaycrude dna solutionwhole genome amplification 