BACKGROUND
genetically engineered mice  have played an essential part in elucidating the functions of specific genes, the mechanisms of disease, embryogenesis and differentiation over recent decades.
although massive numbers of gem have been generated and analyzed throughout the world  <cit> , there is still a demand for more efficient methods of generating de novo gem.
genome editing using programmable nucleases, such as zinc-finger nucleases , transcription activator-like effector nucleases , and clustered regularly interspaced short palindromic repeats / crispr-associated protein  <dig> , is an easy and efficient strategy to generate gem  <cit> .
in particular, crispr/cas <dig> provides the most convenient method by which to produce gem because of its simple construction and high dna double-strand break -inducing activity  <cit> .
a conventional crispr/cas <dig> system consists of cas <dig> nuclease and a single guide rna  that targets a specified genomic locus containing a 20-base sequence defined by the grna and a protospacer adjacent motif  sequence defined by cas <dig>  the complex then cleaves the double-stranded dna.
dsbs are mainly repaired by error-prone non-homologous end-joining , randomly inducing insertions and/or deletions, which cause targeted gene disruption.
the conventional crispr/cas <dig> complex does not form a dimer, unlike zfns and talens, and it often leads to off-target mutations with high frequency  <cit> .
although the frequencies of off-target mutations in animal embryos, especially in mice and rats, are reportedly not so high  <cit> , they do exist and therefore present a potential risk even in animals.
to improve the specificity, two derivative technologies have been developed; double-nicking using cas <dig> nickase  <cit>  and foki-dimerization using nuclease-deficient cas <dig> fused to foki   <cit> .
in both strategies, paired grnas are used to recruit two molecules of cas <dig> nickase or foki-dcas <dig> on juxtaposed positions of the target genomic locus, resulting in dna cleavage.
importantly, single grna-guided cas <dig> nickase can only induce a nick, which is less mutagenic than a dsb.
furthermore, single grna-guided foki-dcas <dig> does not damage the genomic dna at all.
therefore, off-target mutations are significantly reduced in these two derivative strategies.
to generate knockout mice using the crispr/cas <dig> system, cas <dig> mrna and grna are generally synthesized using in vitro transcription and used for microinjection  <cit> .
however, mashiko and colleagues described a simplified protocol using pronuclear microinjection of circular plasmid expressing cas <dig> and a grna  <cit> .
direct use of plasmids avoids the need for laborious in vitro transcription and purification steps, and plasmids offer high stability compared with rnas enabling the robust production of knockout mice.
in addition, we recently established an all-in-one crispr/cas <dig> vector system for the assembly of multiple grna cassettes and a cas <dig> nuclease/nickase cassette in a single vector  <cit> .
when combined with these procedures, efficient production of knockout mice mediated by paired grna-guided crispr systems has been thought to be achieved.
regarding the preparation of oocytes for microinjection to generate knockout mice with genome editing technologies, fertilized oocytes were usually collected by mating a male mouse and a superovulated female mouse.
however, preparing fresh fertilized oocytes for every experiment is time-consuming and laborious work.
we have previously established a procedure of using freeze-thawed fertilized oocytes created by in vitro fertilization  for the talen-mediated generation of knockout mice  <cit> , as well as previous reports of transgenic mouse production  <cit> .
in addition, li and colleagues recently reported that fresh fertilized oocytes created by ivf could be used for conventional crispr/cas9-mediated mouse genome editing  <cit> .
however, han and colleagues reported that they failed to produce genome edited mice by pronuclear injection of crispr/cas <dig> plasmid, while cytoplasmic injection of cas <dig> mrna and purified grna resulted in successful genome editing  <cit> .
thus, the applicability of cryopreservation and ivf technologies for various crispr systems is another important advance in establishing the streamlined crispr/cas <dig> plasmid injection-mediated production of knockout mice.
here, we expand the multiplex grna assembly system to include foki-dcas <dig>  and construct cas <dig> nuclease-, cas <dig> nickase- and foki-dcas9-type vectors for the generation of knockout mice.
the grnas for each system are designed to target the same locus of an endogenous gene, and comparisons of the birth rate and the mutation rate are conducted.
the applicability of cryopreservation and ivf is also investigated.
we further examined whether pronuclear injection of a single all-in-one plasmid expressing foki-dcas <dig> and four grnas could achieve multiplex genome editing in mouse embryos.
RESULTS
construction of an all-in-one vector system for foki-dcas9-mediated genome editing
we previously established a system for creating all-in-one crispr/cas <dig> vectors expressing cas <dig> nuclease or nickase and up to seven grnas  <cit> , and have distributed these plasmids as a “multiplex crispr/cas <dig> assembly system kit” via addgene .
to expand the system for foki-dcas9-mediated genome editing, we first created px330a_foki-1x vectors.
using these newly constructed plasmids and px330s- vectors included in the addgene kit, we can create an all-in-one vector for foki-dcas9-mediated genome editing, not only for single but also for multiplex gene editing .
the new vectors will expectedly be distributed by addgene as a supplemental package of the current kit .figure  <dig> schematic illustration of the all-in-one foki-dcas <dig> vector construction system.
single grna-expressing vectors are constructed by the insertion of annealed oligonucleotides.
subsequently, the grna cassettes are assembled using golden gate cloning.
amp, ampicillin; spec, spectinomycin; u <dig>  human u <dig> promoter; cbh, chicken beta-actin short promoter.
design of crispr/cas <dig> nuclease, cas <dig> nickase, and foki-dcas <dig> vectors to target the interleukin- <dig>  gene
to compare the applicability of the three crispr/cas <dig> systems with cas <dig> nuclease, cas <dig> nickase, or foki-dcas <dig> in mouse genome editing, we designed and constructed crispr/cas <dig> vectors as shown in figure  <dig>  although the three systems require different design parameters and it is difficult to compare the genome editing efficacy with exactly the same grnas, we tried to target almost the same genomic region, which is around exon  <dig> of the il <dig> gene.
for cas <dig> nuclease, two grnas, grna_a and grna_b, were designed within the exon and oligonucleotides for these grnas were cloned separately .
for cas <dig> nickase, grna_b and grna_c were designed with a 7-bp offset .
when inducing double nick-mediated genome modification, the optimal range of the offset length is around 0–10 bp  <cit> , whereas foki-dcas <dig> requires 13- to 18-bp offsets for highly-efficient targeted mutagenesis  <cit> .
thus, we designed another grna, grna_d, in combination with grna_b for foki-dcas <dig> .figure  <dig> the target sequence and the constructed vectors.
the genomic region around exon  <dig> of the il <dig> gene was targeted by cas <dig> nuclease , cas <dig> nickase , or foki-dcas <dig>  vectors.
the target sequence of each grna is indicated by colored bases.
the pam sequence is underlined.
activity validation of the all-in-one foki-dcas <dig> vector
since the genome editing potential of the all-in-one vector containing the foki-dcas <dig> cassette was unknown, we evaluated the activity of the foki-dcas9_bd vector in mouse embryos.
the foki-dcas9_bd vector was microinjected into the pronucleus of mouse fertilized oocytes.
twenty-one survival oocytes were cultured for  <dig>  days.
we observed seven blastocysts, and six of these embryos developed into expanded blastocysts.
each blastocyst was collected into a microtube, and then pcr was performed to amplify the targeted locus.
we verified that at least one mutation had been induced in three embryos by direct sequencing .
in addition, bacterial cloning of the pcr products followed by dna sequencing revealed high mutation rates  , confirming the powerful potential of the all-in-one foki-dcas <dig> vector for gem generation.figure  <dig> sequence analysis of blastocysts injected with the foki-dcas9_bd vector.
the pcr products were analyzed by direct sequencing , followed by sequencing of subcloned plasmids .
the wild-type il <dig> sequence is shown at the top  with the grna target sequences .
the pam sequence is enclosed in red boxes.
deletions are indicated by dashes and substitutions and insertions are enclosed in a black box.
a blue box indicates the partial sequence of exon  <dig>  only one or two types of mutations were detected in direct sequencing , whereas various mutation patterns were observed in subcloned sequencing .
numbers in red letters on the right side of each sequence indicate the frequencies of the corresponding alleles .
gem generation by microinjection of all-in-one crispr/cas <dig> plasmids into freeze-thawed fertilized oocytes
we next conducted microinjection of the three types of crispr/cas <dig> vectors into freeze-thawed fertilized oocytes for efficient and convenient genome engineering in mice.
using reproductive engineering techniques, such as ivf and freeze-thawing, fertilized oocytes could be obtained with high efficiency .
each crispr/cas <dig> vector was directly injected into the pronucleus of freeze-thawed fertilized oocytes prepared by mating or ivf.
the survival oocytes after microinjection were transferred into the oviducts of pseudopregnant icr female mice.
all the surrogate female mice gave birth to pups , and all the pups excluding one pup died from cannibalism were genotyped by direct sequencing analysis .
although we have shown that the combinatorial screening strategy is required for complete genotyping  <cit> , direct sequencing is reportedly able to screen mutant mice with high mutation rates  <cit> .table  <dig>
generation of
il11
mutants using freeze-thawed fertilized oocytes
method
vector
injected
survived
transferred
pups
analyzed
mutants
numbers in brackets represent percentages.
*fertilized oocytes were collected from females at 8 weeks of age, whereas 5-week-old females were used for the other injections that oocytes were fertilized by mating.
sequencing analysis revealed that all three types of crispr/cas <dig> vectors could produce mutant mice, but the birth rates and mutation rates of each method were not comparable.
in the cas <dig> nuclease group, the birth rates were relatively low , whereas the mutation rates were high .
conversely, more pups were born in the cas <dig> nickase group , while the number of mutants were fewer than in the cas <dig> nuclease group .
interestingly, the foki-dcas <dig> group exhibited moderate birth rates  and mutation rates .
to confirm the superiority of foki-dcas9-mediated method, we additionally injected the foki-dcas9_bd vector into freeze-thawed fertilized oocytes collected from mature female mice  mated with male mice.
consistent with the results using the oocytes collected from immature female mice , the birth rate and the mutation rate were sufficiently high .
these results suggest not only the robustness of foki-dcas9-mediated mouse genome editing, but also the applicability of oocytes from female mice at various weeks of age, as shown previously using talens  <cit> .
although these results do not necessarily reflect the general properties of the three crispr/cas <dig> systems, they represent an important practical example of crispr/cas9-mediated gem generation.
importantly, the low birth rates of cas <dig> nuclease plasmid-injected mice are comparable with previous studies using inbred strains  <cit> , although high birth rates can be achieved using b6d2f <dig>  hybrid mice  <cit> .
we thus concluded that foki-dcas <dig> plasmid injection might be a powerful strategy for generating knockout mice, especially for inbred strains.
transgene analysis and off-target analysis
since plasmid dna was used for microinjection, we investigated whether dna vectors were integrated into the genome.
genomic pcr of the coding sequence of cas <dig> variants and foki was performed to detect genomic integrants.
in the two founders generated by the injection of nuclease_b vector, the cas <dig> gene fragment was amplified .
altogether, 10%  of the mutant mice carried the transgene.
although the observed transgenicity is slightly higher than in mashiko’s report   <cit> , this may not be a major issue because the transgene can be removed by mating before expansion of the mutant strain.
finally, potential off-target sites for each grna target site were analyzed by direct sequencing.
the top three candidates for each site were selected using the crispr design tool  .
we then sequenced all of the  <dig> founders , but no off-target mutations were detected.
however, our analysis and all of the previously reported off-target analyses in mice provide limited genomic context, and the potential risk of off-target mutagenesis in disparate parts of the genome is undeniable.
in fact, tsai and colleagues recently revealed that crispr/cas <dig> can induce various genomic rearrangements including megabase-scale large deletions and chromosomal translocations in cultured cells  <cit> .
they also showed that most of these experimentally demonstrated off-target sites were not identified as off-target candidates using any computational prediction tool including the crispr design tool.
also in mice, these concealed off-target effects can exist, but they are difficult to detect because they cannot be predicted by in silico analysis.
we thus believe that a highly specific gene targeting strategy, such as foki-dcas <dig>  is important not only for cultured cell applications but also for the creation of gem.
multiplex genome editing in mouse embryos using a single all-in-one foki-dcas <dig> vector
we finally examined whether multiple gene targeting could be applied using microinjection of a single all-in-one foki-dcas <dig> vector simultaneously expressing four grnas and foki-dcas <dig>  we designed grnas to target exon  <dig> of regenerating islet-derived  <dig> beta  and regenerating islet-derived  <dig> gamma  genes, which are located in the chromosome  <dig> with  <dig> -kb distance .
the all-in-one foki-dcas <dig> vector was microinjected into the pronucleus of fertilized oocytes.
twenty-eight survival oocytes were cultured for  <dig>  days, and nine embryos developed into expanded blastocysts.
each blastocyst was collected into a microtube, and then pcr was performed to amplify around the both target loci.
direct sequencing analyses revealed that more than 50% of the embryos possessed mutated alleles in the both target loci  .
more importantly, five embryos  possessed mutations in the both genes, showing high potential for creating double knockout mice mediated by microinjection of all-in-one foki-dcas <dig> vector.
we further performed out-out pcr to investigate whether chromosomally deleted alleles exist, but no intended amplicons were observed using blastocyst pcr genotyping.
further examinations are needed to clarify the possible existence of chromosomally deleted alleles in pups.figure  <dig> schematic design of multiple gene targeting using all-in-one foki-dcas <dig> vector.
the genomic context around the targeted loci.
the target sites are located in exon  <dig> of reg3b and reg3g genes.
the target sequence and the constructed vectors.
the target sequence of each grna is indicated by colored bases.
the pam sequence is underlined.
and reg3g
loci.
the wild-type sequence of reg3b and reg3g are shown at the top  with the grna target sequences .
the pam sequence is enclosed in red boxes.
deletions are indicated by dashes.
blastocyst numbers on the left side of each sequence in  and  are identical.
most blastocysts had multiple types of mutations at the reg3b locus.
the wild-type reg3b sequence was not detected in blastocyst # <dig> and # <dig>  in the other blastocysts, the wild-type reg3b sequence was detected along with mutant sequence.
regarding reg3g locus, the wild-type sequence was detected in all blastocysts with mutant sequence.
CONCLUSIONS
our study compared the genome editing efficiency in one-cell mouse embryos microinjected with various crispr/cas <dig> vectors expressing grna and cas <dig> nuclease, cas <dig> nickase, or foki-dcas <dig>  constructed by an all-in-one vector system.
in addition, we demonstrated the applicability of ivf and freeze-thawing of oocytes in gem generation mediated by the methods described above, which makes preparation of fertilized oocytes more convenient.
our findings suggest that the foki-dcas9-mediated approach is a highly reliable strategy for creating gem, including double knockout mice, and we provide accessible platforms for the crispr/cas9-based generation of gem.

 genetically engineered mice have played an essential part in elucidating the functions of specific genes, the mechanisms of disease, embryogenesis and differentiation over recent decades. although massive numbers of gem have been generated and analyzed throughout the world, there is still a demand for more efficient methods of generating de novo gem. genome editing using programmable nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats / crispr-associated protein, is an easy and efficient strategy to generate gem. in particular, crispr/cas provides the most convenient method by which to produce gem because of its simple construction and high dna double-strand break -inducing activity. a conventional crispr/cas system consists of cas nuclease and a single guide rna that targets a specified genomic locus containing a 20-base sequence defined by the grna and a protospacer adjacent motif sequence defined by cas the complex then cleaves the double-stranded dna. dsbs are mainly repaired by error-prone non-homologous end-joining, randomly inducing insertions and/or deletions, which cause targeted gene disruption. the conventional crispr/cas complex does not form a dimer, unlike zfns and talens, and it often leads to off-target mutations with high frequency. although the frequencies of off-target mutations in animal embryos, especially in mice and rats, are reportedly not so high, they do exist and therefore present a potential risk even in animals. to improve the specificity, two derivative technologies have been developed; double-nicking using cas nickase and foki-dimerization using nuclease-deficient cas fused to foki. in both strategies, paired grnas are used to recruit two molecules of cas nickase or foki-dcas on juxtaposed positions of the target genomic locus, resulting in dna cleavage. importantly, single grna-guided cas nickase can only induce a nick, which is less mutagenic than a dsb. furthermore, single grna-guided foki-dcas does not damage the genomic dna at all. therefore, off-target mutations are significantly reduced in these two derivative strategies. to generate knockout mice using the crispr/cas system, cas mrna and grna are generally synthesized using in vitro transcription and used for microinjection. however, mashiko and colleagues described a simplified protocol using pronuclear microinjection of circular plasmid expressing cas and a grna. direct use of plasmids avoids the need for laborious in vitro transcription and purification steps, and plasmids offer high stability compared with rnas enabling the robust production of knockout mice. in addition, we recently established an all-in-one crispr/cas vector system for the assembly of multiple grna cassettes and a cas nuclease/nickase cassette in a single vector. when combined with these procedures, efficient production of knockout mice mediated by paired grna-guided crispr systems has been thought to be achieved. regarding the preparation of oocytes for microinjection to generate knockout mice with genome editing technologies, fertilized oocytes were usually collected by mating a male mouse and a superovulated female mouse. however, preparing fresh fertilized oocytes for every experiment is time-consuming and laborious work. we have previously established a procedure of using freeze-thawed fertilized oocytes created by in vitro fertilization for the talen-mediated generation of knockout mice, as well as previous reports of transgenic mouse production. in addition, li and colleagues recently reported that fresh fertilized oocytes created by ivf could be used for conventional crispr/cas9-mediated mouse genome editing. however, han and colleagues reported that they failed to produce genome edited mice by pronuclear injection of crispr/cas plasmid, while cytoplasmic injection of cas mrna and purified grna resulted in successful genome editing. thus, the applicability of cryopreservation and ivf technologies for various crispr systems is another important advance in establishing the streamlined crispr/cas plasmid injection-mediated production of knockout mice. here, we expand the multiplex grna assembly system to include foki-dcas and construct cas nuclease-, cas nickase- and foki-dcas9-type vectors for the generation of knockout mice. the grnas for each system are designed to target the same locus of an endogenous gene, and comparisons of the birth rate and the mutation rate are conducted. the applicability of cryopreservation and ivf is also investigated. we further examined whether pronuclear injection of a single all-in-one plasmid expressing foki-dcas and four grnas could achieve multiplex genome editing in mouse embryos.  construction of an all-in-one vector system for foki-dcas9-mediated genome editing we previously established a system for creating all-in-one crispr/cas vectors expressing cas nuclease or nickase and up to seven grnas, and have distributed these plasmids as a “multiplex crispr/cas assembly system kit” via addgene. to expand the system for foki-dcas9-mediated genome editing, we first created px330a_foki-1x vectors. using these newly constructed plasmids and px330s- vectors included in the addgene kit, we can create an all-in-one vector for foki-dcas9-mediated genome editing, not only for single but also for multiplex gene editing. the new vectors will expectedly be distributed by addgene as a supplemental package of the current kit.figure schematic illustration of the all-in-one foki-dcas vector construction system. single grna-expressing vectors are constructed by the insertion of annealed oligonucleotides. subsequently, the grna cassettes are assembled using golden gate cloning. amp, ampicillin; spec, spectinomycin; u human u promoter; cbh, chicken beta-actin short promoter. design of crispr/cas nuclease, cas nickase, and foki-dcas vectors to target the interleukin- gene to compare the applicability of the three crispr/cas systems with cas nuclease, cas nickase, or foki-dcas in mouse genome editing, we designed and constructed crispr/cas vectors as shown in figure  although the three systems require different design parameters and it is difficult to compare the genome editing efficacy with exactly the same grnas, we tried to target almost the same genomic region, which is around exon of the il gene. for cas nuclease, two grnas, grna_a and grna_b, were designed within the exon and oligonucleotides for these grnas were cloned separately. for cas nickase, grna_b and grna_c were designed with a 7-bp offset. when inducing double nick-mediated genome modification, the optimal range of the offset length is around 0–10 bp, whereas foki-dcas requires 13- to 18-bp offsets for highly-efficient targeted mutagenesis. thus, we designed another grna, grna_d, in combination with grna_b for foki-dcas.figure the target sequence and the constructed vectors. the genomic region around exon of the il gene was targeted by cas nuclease, cas nickase, or foki-dcas vectors. the target sequence of each grna is indicated by colored bases. the pam sequence is underlined. activity validation of the all-in-one foki-dcas vector since the genome editing potential of the all-in-one vector containing the foki-dcas cassette was unknown, we evaluated the activity of the foki-dcas9_bd vector in mouse embryos. the foki-dcas9_bd vector was microinjected into the pronucleus of mouse fertilized oocytes. twenty-one survival oocytes were cultured for  days. we observed seven blastocysts, and six of these embryos developed into expanded blastocysts. each blastocyst was collected into a microtube, and then pcr was performed to amplify the targeted locus. we verified that at least one mutation had been induced in three embryos by direct sequencing. in addition, bacterial cloning of the pcr products followed by dna sequencing revealed high mutation rates, confirming the powerful potential of the all-in-one foki-dcas vector for gem generation.figure sequence analysis of blastocysts injected with the foki-dcas9_bd vector. the pcr products were analyzed by direct sequencing, followed by sequencing of subcloned plasmids. the wild-type il sequence is shown at the top with the grna target sequences. the pam sequence is enclosed in red boxes. deletions are indicated by dashes and substitutions and insertions are enclosed in a black box. a blue box indicates the partial sequence of exon only one or two types of mutations were detected in direct sequencing, whereas various mutation patterns were observed in subcloned sequencing. numbers in red letters on the right side of each sequence indicate the frequencies of the corresponding alleles. gem generation by microinjection of all-in-one crispr/cas plasmids into freeze-thawed fertilized oocytes we next conducted microinjection of the three types of crispr/cas vectors into freeze-thawed fertilized oocytes for efficient and convenient genome engineering in mice. using reproductive engineering techniques, such as ivf and freeze-thawing, fertilized oocytes could be obtained with high efficiency. each crispr/cas vector was directly injected into the pronucleus of freeze-thawed fertilized oocytes prepared by mating or ivf. the survival oocytes after microinjection were transferred into the oviducts of pseudopregnant icr female mice. all the surrogate female mice gave birth to pups, and all the pups excluding one pup died from cannibalism were genotyped by direct sequencing analysis. although we have shown that the combinatorial screening strategy is required for complete genotyping, direct sequencing is reportedly able to screen mutant mice with high mutation rates.table generation of il11 mutants using freeze-thawed fertilized oocytes method vector injected survived transferred pups analyzed mutants numbers in brackets represent percentages. *fertilized oocytes were collected from females at 8 weeks of age, whereas 5-week-old females were used for the other injections that oocytes were fertilized by mating. sequencing analysis revealed that all three types of crispr/cas vectors could produce mutant mice, but the birth rates and mutation rates of each method were not comparable. in the cas nuclease group, the birth rates were relatively low, whereas the mutation rates were high. conversely, more pups were born in the cas nickase group, while the number of mutants were fewer than in the cas nuclease group. interestingly, the foki-dcas group exhibited moderate birth rates and mutation rates. to confirm the superiority of foki-dcas9-mediated method, we additionally injected the foki-dcas9_bd vector into freeze-thawed fertilized oocytes collected from mature female mice mated with male mice. consistent with the results using the oocytes collected from immature female mice, the birth rate and the mutation rate were sufficiently high. these results suggest not only the robustness of foki-dcas9-mediated mouse genome editing, but also the applicability of oocytes from female mice at various weeks of age, as shown previously using talens. although these results do not necessarily reflect the general properties of the three crispr/cas systems, they represent an important practical example of crispr/cas9-mediated gem generation. importantly, the low birth rates of cas nuclease plasmid-injected mice are comparable with previous studies using inbred strains, although high birth rates can be achieved using b6d2f hybrid mice. we thus concluded that foki-dcas plasmid injection might be a powerful strategy for generating knockout mice, especially for inbred strains. transgene analysis and off-target analysis since plasmid dna was used for microinjection, we investigated whether dna vectors were integrated into the genome. genomic pcr of the coding sequence of cas variants and foki was performed to detect genomic integrants. in the two founders generated by the injection of nuclease_b vector, the cas gene fragment was amplified. altogether, 10% of the mutant mice carried the transgene. although the observed transgenicity is slightly higher than in mashiko’s report, this may not be a major issue because the transgene can be removed by mating before expansion of the mutant strain. finally, potential off-target sites for each grna target site were analyzed by direct sequencing. the top three candidates for each site were selected using the crispr design tool. we then sequenced all of the founders, but no off-target mutations were detected. however, our analysis and all of the previously reported off-target analyses in mice provide limited genomic context, and the potential risk of off-target mutagenesis in disparate parts of the genome is undeniable. in fact, tsai and colleagues recently revealed that crispr/cas can induce various genomic rearrangements including megabase-scale large deletions and chromosomal translocations in cultured cells. they also showed that most of these experimentally demonstrated off-target sites were not identified as off-target candidates using any computational prediction tool including the crispr design tool. also in mice, these concealed off-target effects can exist, but they are difficult to detect because they cannot be predicted by in silico analysis. we thus believe that a highly specific gene targeting strategy, such as foki-dcas is important not only for cultured cell applications but also for the creation of gem. multiplex genome editing in mouse embryos using a single all-in-one foki-dcas vector we finally examined whether multiple gene targeting could be applied using microinjection of a single all-in-one foki-dcas vector simultaneously expressing four grnas and foki-dcas we designed grnas to target exon of regenerating islet-derived beta and regenerating islet-derived gamma genes, which are located in the chromosome with -kb distance. the all-in-one foki-dcas vector was microinjected into the pronucleus of fertilized oocytes. twenty-eight survival oocytes were cultured for  days, and nine embryos developed into expanded blastocysts. each blastocyst was collected into a microtube, and then pcr was performed to amplify around the both target loci. direct sequencing analyses revealed that more than 50% of the embryos possessed mutated alleles in the both target loci. more importantly, five embryos possessed mutations in the both genes, showing high potential for creating double knockout mice mediated by microinjection of all-in-one foki-dcas vector. we further performed out-out pcr to investigate whether chromosomally deleted alleles exist, but no intended amplicons were observed using blastocyst pcr genotyping. further examinations are needed to clarify the possible existence of chromosomally deleted alleles in pups.figure schematic design of multiple gene targeting using all-in-one foki-dcas vector. the genomic context around the targeted loci. the target sites are located in exon of reg3b and reg3g genes. the target sequence and the constructed vectors. the target sequence of each grna is indicated by colored bases. the pam sequence is underlined. and reg3g loci. the wild-type sequence of reg3b and reg3g are shown at the top with the grna target sequences. the pam sequence is enclosed in red boxes. deletions are indicated by dashes. blastocyst numbers on the left side of each sequence in and are identical. most blastocysts had multiple types of mutations at the reg3b locus. the wild-type reg3b sequence was not detected in blastocyst # and # in the other blastocysts, the wild-type reg3b sequence was detected along with mutant sequence. regarding reg3g locus, the wild-type sequence was detected in all blastocysts with mutant sequence.  our study compared the genome editing efficiency in one-cell mouse embryos microinjected with various crispr/cas vectors expressing grna and cas nuclease, cas nickase, or foki-dcas constructed by an all-in-one vector system. in addition, we demonstrated the applicability of ivf and freeze-thawing of oocytes in gem generation mediated by the methods described above, which makes preparation of fertilized oocytes more convenient. our findings suggest that the foki-dcas9-mediated approach is a highly reliable strategy for creating gem, including double knockout mice, and we provide accessible platforms for the crispr/cas9-based generation of gem. 
we constructed three types of crispr/cas vectors expressing: 1) single guide rna and cas nuclease, 2) two grnas and cas nickase, and 3) two grnas and foki-dcas targeting the same genomic locus. cas nuclease resulted in the highest mutation rates with the lowest birth rates, while cas nickase resulted in the highest birth rates with the lowest mutation rates. foki-dcas presented well-balanced mutation and birth rates. although we previously demonstrated the applicability of oocyte cryopreservation technologies and various genotyping methods in the production of transcription activator-like effector nuclease-mediated genome editing in mice, it has not yet been clarified whether these techniques can be applied to the crispr/cas9-mediated generation of knockout mice. in this study, we investigated easy, efficient, and robust methods of creating knockout mice using several crispr/cas systems. our report offers several choices of researcher-friendly consolidated procedures for making crispr/cas9-mediated knockout mice, with sophisticated construction systems for various types of crispr vectors, convenient preparation of in vitro fertilized or mated freeze-thawed oocytes, and an efficient method of mutant screening.
 clustered regulatory interspaced short palindromic repeats /crispr-associated protein -mediated genome editing permits the rapid production of genetically engineered mice. to make the most of this innovative technology, a streamlined procedure is needed for the robust construction of crispr/cas vectors, the efficient preparation of mouse oocytes, and refined genotyping strategies. although we previously demonstrated the applicability of oocyte cryopreservation technologies and various genotyping methods in the production of transcription activator-like effector nuclease-mediated genome editing in mice, it has not yet been clarified whether these techniques can be applied to the crispr/cas9-mediated generation of knockout mice. in this study, we investigated easy, efficient, and robust methods of creating knockout mice using several crispr/cas systems.  we constructed three types of crispr/cas vectors expressing: 1) single guide rna and cas nuclease, 2) two grnas and cas nickase, and 3) two grnas and foki-dcas targeting the same genomic locus. these vectors were directly microinjected into the pronucleus of freeze-thawed fertilized oocytes, and surviving oocytes were transferred to pseudopregnant icr mice. cas nuclease resulted in the highest mutation rates with the lowest birth rates, while cas nickase resulted in the highest birth rates with the lowest mutation rates. foki-dcas presented well-balanced mutation and birth rates. furthermore, we constructed a single all-in-one foki-dcas vector targeting two different genomic loci, and validated its efficacy by blastocyst analysis, resulting in highly efficient simultaneous targeted mutagenesis.  our report offers several choices of researcher-friendly consolidated procedures for making crispr/cas9-mediated knockout mice, with sophisticated construction systems for various types of crispr vectors, convenient preparation of in vitro fertilized or mated freeze-thawed oocytes, and an efficient method of mutant screening. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords knockout mousepronuclear microinjectionin vitro fertilizationfreeze-thawingcrispr/cas9double-nickingfoki-dcas9issue-copyright-statement© the author 2015 