- The genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis. - Here, we present a clone-based study design that has allowed a detailed investigation of off-target and de novo mutagenesis in a cattle line bearing edits in the PMEL gene for diluted coat-colour.. - Furthermore, an in-depth analysis of de novo mutations across the edited and non-edited cloned calves revealed that the mutation frequency and spectra were unaffected by editing status. - Cells in culture, however, appeared to have a distinct mutation signature where de novo mutations were predominantly C >. - Conclusions: We found no detectable CRISPR-Cas9 associated off-target mutations in the gene-edited cells or calves derived from the gene-edited cell line. - Comparison of de novo mutation in two gene-edited calves and three non-edited control calves did not reveal a higher mutation load in any one group, gene-edited or control, beyond those anticipated from spontaneous mutagenesis. - Full list of author information is available at the end of the article. - The poten- tial for off-target mutations have been associated with non-unique matches and sequence mismatches distal from the PAM sequences at the 5’ end of the gRNA [4–. - Whole genome sequencing (WGS) is a less biased approach to off-target mutation detection and enables analysis of single nucleotide vari- ants (SNV), small insertions and deletions (indels), and some structural variants (SV), that may arise as a result of the use of CRISPR-Cas9 mediated gene-editing.. - However, since cells naturally accumulate de novo muta- tions through spontaneous mutagenesis during cell div- ision, it is challenging to distinguish mutations attributable to the application of gene-editing technolo- gies from those that occur spontaneously. - To character- ise any off-target mutagenesis, one approach is to quantify changes in detectable de novo mutation be- tween gene-edited samples and controls, and then assess whether candidate variants do, or do not, sit in biologic- ally plausible off-target sites. - [15] used a trio-based study design to investigate off-target effects of CRISPR-Cas9 and showed that the off-target mutation rate was negligible and the de novo mutation rate in edi- ted animals was comparable to their non-edited controls.. - In this study, we conduct the first WGS analysis in cloned cattle generated from a gene-edited cell line to evaluate off-target events and de novo mutagenesis asso- ciated with the application of CRISPR-Cas9 mediated gene-editing and cloning to create live cattle for use in agriculture. - Taking advantage of the clone-based study design, we used WGS and other molecular approaches to comprehen- sively screen for off-target SNVs, indels, and SVs that could be attributed to the use of CRISPR-Cas9 mediated gene-editing. - We found no detectable CRISPR-Cas9 as- sociated off-target mutations, and that the de novo. - Origin of the study material and analysis of whole genome sequence data. - This reduced the total number of candidates for variants induced by potential off-target events or spontaneous de novo mutagenesis to 457.. - A subset of the male primary bovine fetal fibroblasts (BEF2) were transfected with a plasmid-encoded, PMEL-specific editor and a single stranded homology directed repair (HDR) template. - The remaining 218 candidate off-target/de novo mutations were then manually examined by visualisation of se- quence reads in the Integrative Genomics Viewer (IGV). - We found 230 (of variants that mapped within 50 bp of the 1,166 candidate off-target sites. - 2 Filtering criteria applied to raw variant calls to identify potential off-target mutations and spontaneous de novo mutations in the gene- edited cell line CC14. - *Variants were kept if also present in 1805 and/or B071 **Predicted heterozygous de novo mutations were also filtered for their presence in calves 1805 and B071. - filtered out due to poor read quality, one captured the on-target mutation at the edited site, and one site was called in the sample of the non-edited control calf 1803.. - Long molecule sequencing of the on-target site. - Alignment of the long reads to the PMEL-specific CRISPR-Cas9 expression plasmid sequence using minimap2 [24] revealed no matches, suggesting that the editing plasmid was un- likely to have integrated at the on-target site.. - A de novo assembly of these reads indicated that these reads could not be assembled into a single contiguous sequence, and alignment to the bovine genome using BLAST [26] did not highlight any sequence overlap.. - Analysis of de novo mutations in the cloned calves The cloned calves used for this study were generated by somatic cell nuclear transfer with donor cells from either the parental cell line BEF2, or the gene-edited cell clone CC14 [20]. - To identify de novo mutations carried by each cloned calf, either originating from the donor cell or occurring during development of the calf, we applied the filtering criteria outlined in Fig. - To differentiate between de novo mutations that likely occurred in cell culture and were subsequently inherited by the cloned. - calves, from de novo mutations that likely occurred dur- ing development of the cloned calves (i.e., after first cell division), we categorised de novo mutations as heterozy- gous or mosaic based on allele dosage at each site (Table 1). - A binomial probability function was applied to determine if the allele dosage at each variant site was consistent with a truly heterozygous genotype expected for a de novo mutation already present in the donor cell.. - When the allele dosage at a variant site was determined to be not statistically different from the expected allele dosage of 0.5, the variant was predicted to be a candidate heterozygous de novo mutation in the cloned calf, whereas if allele dosage was significantly less than 0.5, the variant was predicted to be a candidate mosaic de novo mutation that arose during development of the cloned calf. - All variants were manually assessed in IGV software, after which a proportion of candidate de novo mutations were filtered out due to representing incorrect variant calls, most often due to errors based on proxim- ity to polynucleotide regions, repetitive regions, mis- called variants in other samples, proximity to indels, or misalignment of reads. - Table 1 shows the number of variants that remained after applying the filtering criteria outlined under ‘de novo variants’ in Fig. - de novo’ mutations are those that remained after the manual check.. - Heterozygous de novo mutations. - The majority of de novo mutations present in the cloned calves appear to be heterozygous variants and are likely inherited from the donor cell used for somatic cell nuclear transfer. - A pair- wise comparison of the number of likely heterozygous de novo mutations inherited by each of the cloned calves (Table 1) suggests that the number of mutations ob- served in each clone is statistically different between six of the ten pairs (Table 2). - Based on these results, the number of heterozy- gous de novo mutations inherited by cloned calves gen- erated from the gene-edited cell clone CC14 (1805 and B071) did not appear to be different than those in cloned calves generated from the non-edited, parental cell line BEF and 1804).. - Mosaic de novo mutations. - The number of mosaic de novo mutations identified was more than a magnitude lower than the number of heterozygous de novo muta- tions identified (Table 1). - Table 1 Number of candidate de novo mutations identified after each filter was applied to 31,190 filtered variants across the three control cloned calves and two gene-edited cloned calves. - Heterozygous de novo mutations Mosaic de novo mutations Sample ID Unique to each sample Map quality = 60 Candidate. - de novo. - Likely de novo. - Candidate de novo. - Each ‘likely de novo’ mosaic mutation (Table 1) was manually checked for evidence of a segregating bi-allelic variant on the same read, or read pair, to support the presence of three haplotypes and strengthen the evidence supporting a true mosaic mutation. - Out of the total number of vari- ants predicted to be likely true mosaic mutations: 8/16 variants in variants in 1803, 5/9 variants in 1804, 2/8 variants in 1805, and 5/11 variants in B071 had evidence of three haplotypes and could be con- firmed as true mosaic de novo mutations. - A pair-wise significance test demonstrated that the difference in number of likely mosaic de novo mutations carried by each cloned calf (Table 1) does not appear to be statisti- cally significant, regardless of the cell line of origin (smallest p-value = 0.15 between calves 1802 and 1805;. - These results suggest that the de novo muta- tion rate during embryonic development does not sig- nificantly differ between cloned calves generated using donor cells from a cell clone edited by the CRISPR-Cas9 gene-editing tool, and those generated using a non- edited cell line of the same parental origin.. - Comparison of mosaic de novo mutation rate in clon- ing compared to other reproductive technologies. - We are unaware of any study to date that has attempted to quantify the de novo mutation rate in cloned animals.. - [28] which investigated the number of mosaic de novo mutations reported for generation of animals using other reproductive technologies. - This study used whole genome sequence data from 131 three or four gener- ation pedigrees to investigate de novo mutagenesis in cattle generated via artificial insemination (AI. - Comparison of mosaic de novo mutation in the cloned calves described in this study (n = 5) to that in cattle generated from AI, MOET, and IVF in the Harland et al. - [28] study suggest that the mosaic de novo mutation rate in cloned calves may be. - [28], where increased cell handling and intervention may result in increased incidence of de novo mutagenesis.. - De novo structural variants. - Comparison of de novo mutations between experimental groups. - Comparison of de novo mutation distribution and spectra To further evaluate the candidate de novo mutations across experimental conditions, we categorised muta- tions according to the different stage of their occurrence, and compared mutation distribution and spectra of Table 2 Results (p-values) from two-proportion z-test. - comparing the difference in number of likely heterozygous (top) and mosaic (bottom) de novo mutations observed in the cloned calves. - De novo mutations that arose in cells post plasmid transfection (n = 150. - Table 1) were estimated based on heterozygous de novo mutations in the CC14 cell clone that were sub- sequently inherited by cloned calves B071 and 1805, but absent in all other samples. - The number of de novo mu- tations that emerged during cell clone expansion were estimated based on the sum of mosaic de novo muta- tions in the gene-edited cell clone CC14, and heterozy- gous mutations that were present in any cloned calf, but not in CC14 or the parental cell line, BEF2 (n = 1298;. - The smallest proportion of de novo mutations (n = 58. - Table 1) arose during the development of the cloned calves. - Across the three groups, de novo muta- tions appeared to be randomly dispersed across most of the genome and were not observed to cluster in a group-dependent manner (Fig. - 4a), but a distinct spectra of mutations was observed between de novo mutations that were predicted to have arisen in the cloned calves and those that were predicted to have arisen post plasmid transfection or during cell culture (Fig. - cloned calves (p . - These mutations accounted for 31 % of the total de novo mutations observed in the cloned calves. - We present the first study in cattle based on cloned calves produced by somatic cell nuclear transfer to evaluate unintended off-target mutations, SVs at the on- target site, and unintended integration of the editing plasmid associated with the application of CRISPR-Cas9 mediated gene-editing. - 4 Distribution and spectra of de novo mutations predicted to have arisen in cells post plasmid transfection (red), during the cell culture expansion phase (orange) and during development of the cloned calves (blue). - (b) Proportion of de novo mutations within each mutation class observed between groups. - gene-editing does not affect spontaneous mutagenesis or mutation spectra in subsequent cell divisions post-edit, and de novo mutagenesis in calves derived from the gene-edited cell clone appears to be equivalent to that of controls.. - The number of heterozygous de novo mutations varied significantly between calves, but these changes could not be attributed to any single experimental condition (i.e., cell line, gene editing status, or donor cell origin). - As we expect heterozygous de novo mutations in the cloned calves to have been inherited from their somatic donor. - cells, these results suggest that the use of CRISPR-Cas9 gene-editing is unlikely affecting the expected spontan- eous mutation rate during clonal expansion of the gene- edited cell. - The difference in observed heterozygous de novo mutations may instead be due to differences in the accumulation of unrepaired DNA damage across cells in culture, which could be induced by oxidative damage, damage due to UV exposure, other mutagens, or mech- anical sheer [33, 34]. - Indeed, when we examined the mu- tation spectra for heterozygous de novo mutations, we observed a significant enrichment in C >. - By contrast, the mosaic de novo mutations observed in the cloned calves appeared to be statistically equivalent re- gardless of their edited or non-edited status, although it is important to note that some somatic mutations may not be represented in the WGS data or discarded as se- quencing errors due to their low-abundance or absence in the sampled tissue. - When the frequency of de novo mutations was com- pared to that observed in cattle generated with the as- sistance of reproductive technologies such as AI, MOET and IVF [28], we observed that the average number of de novo mutations reported for the cloned calves was greater than the average number observed in the other groups, but not significantly so when compared to IVF.. - Analysis of the de novo mutation spectra revealed a marked difference in predominant mutation type between the cloned calves and cattle pro- duced by natural matings and other reproductive tech- nologies [38]. - Our results demonstrate that naturally occurring, beneficial genetic variation can be introduced into ani- mals that subsequently show levels of mutagenesis indis- cernible from the de novo mutation rates of un-edited controls. - A major challenge is the ability to detect and quantify off-target mutagenesis above the background de novo mutation rate. - We can identify candidate off-target events using logical filtering criteria and evaluate each site for its biological plausibility based on sequence simi- larity with the on-target site, but it is more difficult to differentiate between off-target mutations and spontan- eous de novo mutations at regions with little homology to the on-target site. - Holstein-Friesian cattle have a baseline spontaneous de novo mutation rate of approxi- mately mutations per bp, per generation [28].. - Spontaneous de novo muta- tions have been observed to follow a typical signature, where there is an expected excess of C >. - Development of sensitive tools that enable accurate detection of genuine off-target events, but also consider natural de novo mutation, may be difficult but will aid to establish the risk profile of gene-editing technologies and ultimately support in- formed consumer decisions.. - Cell clones identified to be homozygous for the targeted 3 bp deletion in exon 1 of the PMEL gene (p.Leu18del. - Identification of off-target mutations. - Investigation of the presence of the PMEL -specific CRISPR- Cas9 expression plasmid. - Identification of de novo mutations. - De novo SNVs and indels unique to each sample were identified using a filtering criteria similar to that de- scribed above for identifying off-target mutations. - From the remaining variants, de novo mutations were identified by the following criteria: (1) keeping heterozygous SNVs and indels specific to each sample. - (3) classifying SNVs and indels as heterozygous or mosaic de novo mutations, where mo- saic variants were defined as having an allele dosage sig- nificantly less than 0.5, as determined by the binomial probability function described previously. - All pair-wise comparisons of de novo mutation rates reported in this study were conducted using a two- proportions Z-test, and comparisons of de novo muta- tion spectra were conducted using Fisher’s exact test.. - GL, ML, RS, DG were involved in supervision of the project. - Off-target Effects in CRISPR/Cas9-mediated Genome Engineering. - De novo assembly of the cattle reference genome with single-molecule sequencing. - Rate of de novo mutation in dairy cattle and potential impact of reproductive technologies
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