Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the method’s success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA–2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.


Abstract:
CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the method's success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA-2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

Introduction:
Defective genes cause several inherited diseases. A better understanding of the mechanisms of these defects is critical to obtaining precise diagnoses and finding new therapeutics. Gene  The recent emergence of genome editing technologies such as ZFN, TALENS and CRISPR-Cas9 enables an improvement in efficiency of gene targeting and has considerably facilitated the generation of genetically-engineered animal models based on homology directed repair of donor constructs in mouse zygotes! [8]. Endonucleases, particularly Class 2 CRISPR systems, generate a precise double strand break (DSB) in the DNA under a chimeric single guide RNA (sgRNA)! [9]. The DSB leads to error-prone, non-homologous end joining (NHEJ) repair or the precise homology-directed repair (HDR) under the guidance of a repair template [8]. In an earlier study, a high success rate (16%) of targeting LoxP sites in cis was reported by using 2 sgRNAs and 2 single-stranded oligonucleotides (ssODN) containing LoxP sites (2sgRNA-2ssODN) flanking a targeted critical exon ( Figure 1) [10].
We sought to investigate the efficiency of the 2sgRNA-2ssODN method for the generation of cKO alleles. We describe here for the first time a global community effort from a consortium of over a dozen laboratories, transgenic core facilities and programs across the world to evaluate the efficiency of generating cKO alleles using the 2sgRNA-2ssODN approach. We surveyed over 50 loci and over 17,000 microinjected or electroporated zygotes using this method, which enabled robust statistical power to evaluate the efficiency of the technique. In contrast to the earlier report [10], we find this method does not efficiently produce cKO alleles. Rather, it generally results in a series of undesired editing events at the cleavage sites which occur nearly 100 fold higher rate than the precise insertion, in cis, of the two LoxP sites.

Material and methods:
Ethical statement: Mecp2 gene targeting using CRISPR-Cas9: Mecp2 left single chimeric guide RNAs (sgRNA) 5'-CCCAAGGATACAGTATCCTA-3' and Mecp2 right sgRNA 5'-AGGAGTGAGGTCTAGTACTT-3' target sites were designed as described in Yang et al [10]. Ultramer Oligonucleotides (Integrated DNA Technologies, Coralville, IA) were designed with sequences to T7 promoter for in vitro transcription, DNA target region, and chimeric RNA sequence. Complimentary oligos for each target sequence were annealed at 95°C for 5 mins and the temperature was reduced 0.20°C/second to 16°C using a PCR machine (BioRad T100) before use as template for sgRNA synthesis. sgRNAs were synthesized with the HiScribe™ T7 Quick High Yield RNA Synthesis Kit (New England Biolabs). Cas9 mRNA was obtained from Life Technologies or in-vitro transcribed from a Chimeric pX330-U6-Chinmeric-BB-CBh-hSpCas9 expression plasmid obtained from Addgene repository (Plasmid 42230; donation from Zhang laboratory).
Cas9 protein was complexed with thesgRNA or crRNA and the trans-activating crRNA [17] and then mixed with the ssODN prior to microinjection. Concentrations and site of injection for Cas9 protein or mRNA, sgRNA, and template repairs for each locus are indicated in Supplementary   Table 1.
Mouse husbandry and zygote microinjection and electroporation: Mice were purchased from various sources and maintained under specific pathogen-free conditions. Mice were maintained under 12/12 hr light cycle and food and water were provided ad libitum. Three to five week-old females were superovulated by intraperitoneal injection of Pregnant Mare Serum Gonadotropin (5IU) followed by intraperitoneal injection of Human Chorionic Gonadotropin hormone (5IU) 48 hours later. Superovulated females were mated with 8 to 20 week-old stud males. The mated females were euthanized the following day and the zygotes were collected from their oviducts. Cytoplasmic or pronuclear injections were performed under an inverted microscope, associated micromanipulators, and a microinjection apparatus.
Electroporation of the embryos were performed with an electroporation device using a cuvette or 1mm plate electrodes with the following parameters: 30 V square wave pulses with 100 ms interval using a BioRad electroporator device or 4 poring pulses (40 V, 3.5 ms, interval 50 ms, 10% voltage decay + polarity) followed by 5 transfer pulses (5!V, 50!ms, interval 50!ms, 40% voltage decay, alternating!+!and!−!polarity) using a NEPA21 electroporator device. Microinjected or electroporated zygotes were either surgically transferred into the ampulla of pseudo-pregnant females or cultured overnight at 37ºC and then surgically transferred at the 2-cell stage of development. Genotyping: DNA extraction was performed on ear punch or tail tip from mouse pups over 15 days using a DNA extraction kit according to the manufacturer instructions. Primers were designed to amplify the regions encompassing the integrated LoxP sequence. PCR was performed using Taq polymerase under standard PCR conditions. The PCR products were then purified with ExoSAP-IT1 or a PCR Clean-Up System kit according to the manufacturer's instructions. Sanger sequencing was performed in core facilities. To identify LoxP insertions, as a general practice at all centers, the two target sites were amplified individually to look for increase in the amplicon size, which occurs if LoxP sites are inserted successfully. If the LoxP insertion was not observed in this first set of PCR analyses, the samples were declared negative, and in many such cases the samples were not analyzed further (as the end goal of the project, ie., generation of floxed allele was not met). In some cases, such samples were also sequenced to assess indels to understand if the guides were successful in cleaving the target site. In some cases, the entire regions encompassing both the guide cleavage sites were amplified to assess for deletions between the cleavage sites.

Statistics:
To determine the statistical differences between proportions or means, we performed a Fisher Exact test or a Kruskal Wallis sum rank test. A Generalized linear model calculation was performed with success of the 2sgRNA-2ssODN method as a response. Predictive variables were: efficiency of the sgRNA, probability of LoxP insertions in 5' and 3 (5'_LoxP and 3'_LoxP), simultaneous insertion of the 2 LoxP sites (interaction between 5'_Loxp and 3'_LoxP) Cas9 mRNA, protein, plasmid and ssODN concentrations and distance between distal and proximal target sites. Variance for each predictor was determined from the diagonal of the variance-covariance matrix. Effect sizes and type II error were determined using Cohen effect size d statistics and power calculation. All statistical analyses were performed using Rstudio v1.1.423. Results were considered statistically significant at p<0.05.

Results:
Mecp2 gene targeting in blastocysts: To assess the efficiency of the technique and compare to previously published results [10], we reproduced an experiment on Mecp2 gene, essential for DNA methylation during development using the same sgRNAs and ssODNs as previously described in the original report [10]. Three independent centers at the Australian National University in Australia (ANU), University of Nebraska Medical Center in the USA (UNMC) and the Czech Centre for Phenogenomics in Czech Republic (IMG) performed these experiments on C57BL/6N inbred strain of mice. We evaluated the success rate of the 2sgRNA-2ssODN method in blastocysts for Mecp2 (Table 1).
Using a concentration mix of 20 ng/µl of Cas9 mRNA, 20 ng/µl of in-vitro transcribed sgRNA, and 10 ng/µl of ssODN, we observed no successful targeting (i.e., correct insertion of 2 LoxP sites in cis configuration) even though both sgRNAs cleaved target DNA as indicated by the presence of indels or integration of a LoxP site at the desired location, which varied from 13% to 34% (Table 1).  Interestingly we noted the occasional presence of mutations within LoxP sites indicating illegitimate repair events at the target site. The frequency of successful targeting of two LoxP sites in cis was previously reported to be 16% [10], which we failed to achieve. One possible explanation is the mouse genetic background influences the likelihood of ssODN integration.
This variance could also be explained by an inherently low probability to successfully replace 2 genomic loci in cis, the lack of efficiency of the sgRNA, or the relatively low sample size.
A global survey of the generation of conditional alleles using 2sgRNA-2ssODN method.
To better understand how to successfully generate conditional alleles using the 2sgRNA-2ssODN approach and to assess its efficiency, we evaluated this method on 56 additional genes and two intergenic regions of the mouse genome from a consortium of 17 institutions across   Table 4). Out of 17,887 (17,557 microinjected and 330 electroporated; see details below) zygotes, 12,764 (71.4%) zygotes were surgically transferred into recipient females. The recipient females gave birth to 1,718 pups (9.6% of the microinjected/electroporated zygotes). As a general practice, at all centers, the mice were first analyzed by PCR to observe the putative insertion of the LoxP sites at both the sites; the animals were declared negative if genotyping did not reveal the presence of the desired allele. In some cases, the loci were further analyzed to assess guide-cleaving activity. Of the 1,684 founder mice, 659 (39%) showed some type of editing (indels and/or substitutions), 235 (14%) and 144 (9%) mice harbored a single LoxP insertion or deletions between the two cleavage sites, respectively ( Figure 2C). The mice for 25 (of the 56) loci were further assessed for additional events including large deletions ( Figure 2C). Of the 487 founder mice analyzed (from those 25 loci), 41%, 11% and 2.7% samples contained indels, single LoxP insertions or large deletions respectively ( Figure 2D). From the 1,684 animals analyzed, only 15 mice (0.87%) were correctly targeted with intact LoxP sites in the cis configuration (Supplementary Table 5 Table 4) from analysis of blastocysts or live mice for 2 out of the 7 loci. In contrast we noted a relatively high frequency of large deletions and indels (up to 39% of large deletions) indicating successful editing. However, none of the loci showed two LoxP sites inserted in cis in the offspring, suggesting that the delivery of CRISPR reagents by electroporation does not make a statistical difference in obtaining a desired outcome from the 2sgRNA-2ssODN floxing approach, although the large numbers of embryos that can be manipulated allows for the recovery of the very small number of those that are correctly targeted.

Discussion
CRISPR-Cas9 technology has greatly facilitated the generation of mouse lines containing knockout or knockin alleles. However, the generation of conditional alleles remains a challenge using traditional ES cells and CRISPR-Cas9 gene editing technologies. An earlier paper demonstrated 16% efficiency with 2 chimeric sgRNAs and 2 single-stranded oligonucleotides to produce conditional alleles in mice [10].
To evaluate the efficiency of this 2sgRNA-2ssODN method, three laboratories replicated the experiments described in the initial report on Mecp2 (10) using the same methods to generate the sgRNA and Cas9 and microinjected the mouse zygotes at similar reagent concentrations.
Although we observed single LoxP site insertions and indels at the cleavage sites, the method was unsuccessful in generating two LoxP sites in cis. A previous report attempting to replicate the findings of Yang et al [10], found an efficiency of floxing Mecp2 varying from 2% to 8% with the 2sgRNA-2ssODN approach [26]. We surmise the lack of efficiency in targeting Mecp2 here was due to a low concentration of reagents delivered by microinjection, a notion corroborated by previous work from Horii and colleagues [26]. Of note, it was reported that up to 6% targeting efficiency was achieved using 25 ng/µl of Cas9, 6 ng/µl of sgRNA and 100 ng/µl of ssODN but toxic to embryonic development; these concentrations are 2-fold higher than those described in Yang et al, 2013 [10].
What determines the success of the 2sgRNA-2ssODN method?
For better understanding the critical factors predicting the likelihood of success with the 2sgRNA-2ssODN approach, we surveyed 56 unique loci in mice zygote. We noted that the efficiency of simultaneous insertion of the two LoxP sites simultaneously was the best predictor of success using this approach. We also noted a low success rate in generating a conditional allele across all centers (< 1%), varying from 0 to 50% (median = 0%) for individual loci. These results are comparable with previous reports demonstrating an important disparity in success rate varying from 0% to 7% of mice harboring two LoxP sites insertions in cis whether delivered by microinjection [26][27][28][29] or by electroporation [26]. We and others also have noted the large number of deletions at the target sites following DNA cleavage [28]. Our results on a larger number of loci suggest the efficiency in generating a successful cKO with the 2sgRNA-2ssODN method is lower than previously described! [10]. One hypothesis for this discrepancy in success rate might relate to strain-specific differences. We analyzed this variable and did not find any significant differences among strains, whether the donor strain was a F1 cross, inbred, or outbred mouse line as a donor strain. Another possibility to improve the efficiency of the method is to avoid recombination between the target sites by placing the LoxP sites hundreds of kb apart. This was reported previously for a success rate varying from 0% to 18% for 6 loci [30]. We did not find such evidence in our data, although our sample size is too small to formally rule out this hypothesis. A recent report found the successful use of sequential introduction of the             -CCTGCTACCCTAACTGCGGT GGG-3' 5'-GGCGGCCCTGACTGAGCGGA GGG-3'   5'-TGCCTATCAAGGGGTAGATCTCACTGAGAGTCATCACCTT  CGCTGAGGGATGCAGCATAGCCTTGGGATCCCACCGAAT  TCATAACTTCGTATAGCATACATTATACGAAGTTATGCAGT  TAGGGTAGCAGGAAGACCGTGGAGTAGTCATGATGCCTG  GATTGAGCCTGGTCCTGGTCCAGGCTAGAGC-3'   5'-AGACAGCCCAGTGTGGAAACACGGGCACCCCTGCTCCG  CTCAGCCTCCCATGAACCCCGGCGGCCCTGACTGAGCAA  GCTTATAACTTCGTATAGCATACATTATACGAAGTTATGGA  GGGTTCCTTAAGAGGCAGCTAGCGCAAGGCTTTGCGTTC  ACATGTACTGTAATAGCCAGTCTTCTAGAGGTG-3