- Background: The development and application of CRISPR technologies for the modification of the genome are rapidly expanding. - With characterization of CpG methylation patterns using whole genome bisulfite sequencing, these CGI methylation disruptions trace the insertion of the donor DNA during the genomic edit. - recombination disrupt the generational methylation pattern stability of the edited CGI within the cells and their cellular lineage within the animal strain, persisting across generations. - modification of the methylome of the affected CGI at the CpG-level.. - Any modification of the methylome of somatic or germline cells could have implications for gene regulation mechanisms governed by the methylation patterns of CGI regions in the application of therapeutic edits of more sensitively regulated genomic regions. - The method described here locates the directed modification of the mouse epigenome that persists over generations. - The pattern is maintained by DNMT1 and associated proteins through cell divisions and during the life of the cell [22]. - Directed reprogramming of the epigenome can replace the epigenetic signatures ac- quired during development, or imposed by the environ- ment [26]. - however, many of the fundamental mechanisms of con- trolling CpG methylation and their impacts are not fully understood. - These changes in the methylome pattern of the genome can induce unexpected changes in the regulation of cellular pathways and characteristics.. - In this study, we highlight perturbations of the epige- nome induced by the incorporation of donor DNA dur- ing CRISPR genome editing. - The result of this sequence replacement or insertion is an alteration of the methyla- tion patterns of CGIs within the region of the genome edit. - This methylome change is linked to the footprint of the inserted donor DNA and is inheritable across gener- ations. - CRISPR edits leveraging HDR mechanisms and donor homology arms that are localized around CGIs modify the methylation patterns of these CGIs, resulting in increased methylation of the CGIs within the region.. - Findings de- scribed within in the methodology here indicate an in- crease in methylation observations within the regions of the adopted homology arms.. - To determine differential methylation of CpG islands across the genomes of the evaluated animals, we se- quenced the genome-wide methylation patterns of CpGs using whole genome bisulfite sequencing (WGBS). - HDR2 028543 CRISPR-mediated HDR insertion of a 2005-bp donor DNA containing eGFP with a poly A signal in an inverted orientation at the start codon of the Rxfp 3 locus. - Strain HDR2 (b) contains a 2005-bp insert and was recombinantly inserted into the Rosa26 locus of the mouse genome using a 1-kb UHA and a 1-kb DHA. - Only a small portion of CGIs in each ani- mal displayed significant epigenetic change from the CGIs of the Control 1 Animal (Table 2) with each ex- perimental comparison closely matching the results of control comparisons. - The distribution of the number of CpG calls observed is shown for HDR1 Animal (a) with CpG locations with ≥ 5 calls, HDR2 Animal (b) with CpG locations with ≥ 5 calls, NHEJ1 Animal (c) with 6,382,801 CpG locations with ≥ 5 calls, Control 1 Animal (d) with CpG locations with ≥ 5 calls, and Control 2 Animal (e) with 8,754,364 CpG locations with ≥ 5 calls. - CRISPR edits mediated by HDR display methylation profiles at CGIs with distinctive increases in methylation Closer observation of the methylome at the edited CGI locations for the edited animals as compared to the con- trol demonstrates the significant deviation in the methy- lation patterns for the HDR modified CGIs. - 5a shows the complete modification of the edited CGI (Chr in the experimental animal (blue squares) beyond the anticipated 20% bio- logical noise seen in the Control 1 Animal (red dia- monds). - The incorporation of the donor DNA homology arms replaced the entire CGI genomic range (gold bar) on each side of the CRISPR-targeted PAM site (dashed line). - Expanding beyond the genomic region influenced by the homology arms of the donor DNA, Fig. - The observed methylation pattern for the CGI of the CRIS PR-edited HDR1 and the Control 1 are illustrated in Fig.. - Figure 6a illustrates the effects of the partial modifica- tion of an edited CGI (Chr . - In this edit, the CRISPR-targeted PAM site (dashed line) is located upstream of the affected CGI.. - During the HDR, only the downstream homology arm was incorporated into the affected CGI range, stretching approximately half the length of the CGI (blue back- ground shading). - The resulting methylation pattern per- turbation is observed in this region of the CGI only. - The downstream portion of the CGI methylome (unshaded background) remains unmodified from that of the con- trol animal. - 6d) demonstrate the ele- vated methylation of the HDR2 CGI with a distinct A. - border of methylation inflection at the interaction site of the terminus of the downstream homology arm.. - In contrast, the edited CGI using NHEJ repair to insert donor DNA retains a methylation pattern that is very similar to the Control 1 Animal, demonstrating that the lack of replacement of the edited CGI (Chr . - The NHEJ repaired CGI region is similar to that of the Control 1 Animal, supporting the CGI falling into the Case 4 category of CGIs in the statistical work- flow (Table 2). - Similarity between NHEJ1 and Control 1 was seen at an expanded view of the genomic region that included 7000 bp upstream and downstream of the CGI. - These observations were reproduced in comparisons of the edited CGIs of modi- fied animals to the unedited CGIs of Control 2 Animal.. - CGIs with statistically relevant methylation changes CGIs containing statistically significant change from that of the control animal and containing changes beyond those considered biological noise were categorized as Case 1 CGIs within the statistical workflow. - Organized in smallest to largest order of p -value, the CRISPR-edit sites using HDR in the HDR1 Animal (3.852E-48) and the HDR2 Animal (5.384E-23) rank as the most significantly methylated CGIs within the ge- nomes of the experimental animals as compared to the Control 1 Animal (Tables 3 and 4. - These data highlight the generational persistence of the epigenetic changes at the edit sites within the progeny. - The degree of methyla- tion distribution is skewed toward the higher percentage of increased methylation in the HDR1 Animal at the re- gion of the edit due to complete CGI methylome modifi- cation. - The genetically-edited CGI of the NHEJ1 Animal using NHEJ repair to introduce an indel did not rank within the most significant Case 1 CGIs for the animal (Table 5), in- stead the edited CGI ranking within the Case 4 category of the comparisons. - The Case 1 CGIs of the NHEJ1 Ani- mal were similar in their measure (Fig. - Supplemental Figures A and B illustrate the profiles of the top two Case 1 CGIs for the NHEJ1 Animal, indicating a non- uniformed distribution of methylation increase unlike the more uniformed increase displayed in the HDR edited sites (Figs. - Further, the comparison of the CGI methylation changes between Control 1 and Con- trol 2 (Table 6) highlights a significantly methylated CGI. - (Chr with similar p -values to those observed in the partially edited CGI of the HDR2 Animal (Tables 4 and 5. - One significant difference between the significantly methylated CGIs of the control comparisons compared to those of the homology-directed repair edited animal is the consistency of the CpG methylation across the CGI. - When visualized, the methylation patterns of the CGIs between Control 1 and Control 2 (Table 6) are in- consistent with CpG methylation calls rising and falling irregularly across the CGIs. - In contrast, the methylation patterns of the edited sites display a uniformed increase of methylation across the range of the incorporated homology arm (Figs. - 5 Methylomic difference of a completely edited CpG island within the genome of the HDR1 Animal using homology-directed repair in a CRISPR-mediated edit. - 4b illustrates the comparison of the modified region to flanking endogenous genomic regions by displaying the variance of methylation patterns for 7000 bp upstream and downstream of the CGI. - The dashed line indicates the protospacer adjacent motif (PAM) location of the targeted CRISPR cut site. - Synthetic homology arms corresponded to genomic sequence 1000 bp upstream and downstream of the PAM site. - The comparison of the percent methylation observed at the localized CGI region for the CRISPR-edited animal (Fig. - 4d) demonstrate the introduced methylation variance as a result of the introduction of the donor DNA. - Throughout the life of the animal, the spatial and temporal modifications of CGI methyla- tion patterns across the genome are critical for tissue- specific differentiation and development [21]. - 6 Methylomic difference of a partially edited CpG island within the genome of the HDR2 Animal using homology-directed repair in a CRIS PR-mediated edit. - yellow bar) within the genome of the HDR2 Animal, resulting in a destabilized methylation pattern (blue squares) as compared to the Control 1 Animal methylation patterns at the same location (red diamonds). - The regions with methylation patterns modified by the incorporation of homology arms within the CGI and upstream of the CGI are shaded in a light blue background. - 5b illustrates the comparison of the modified region to flanking endogenous genomic regions by displaying the variance of methylation patterns for 7000 bp upstream and downstream of the CGI. - The methylation patterns of the unmodified downstream portion of the CGI (unshaded) for the edited CGI as well as the flanking regions of endogenous genome displayed a methylation pattern similar to the control. - The comparison of the percent methylation of the CpG sites observed at the localized CGI region for the CRISPR-edited animal (Fig. - 5d) demonstrates the introduced methylation variance as a result of the adoption of the donor DNA. - In contrast, the NHEJ modified CGI of the NHEJ1 Animal did not display a significant change in methylation pattern compared to the control animal and was not observed in the top ranking chan- ging CGIs, reflecting the lack of integration of donor DNA within the genomic sequence of the edited CGI.. - 7 Methylomic variance of a CpG island within the genome of the NHEJ1 Animal flanking a non-homologous end-joining repair in a CRISPR- mediated edit. - yellow bar) was inserted within the genome of the NHEJ1 Animal, resulting in a methylation pattern of the edited animal (blue squares) that was similar to that of the Control 1 Animal at the same location (red diamonds) localized around the CGI edit site (Fig. - 6a) as well as 7000 bp upstream and downstream of the edit (Fig. - 5d) demonstrates a lack of variance in methylation pattern as a result of non-homologous end-joining insertion of the 48-bp duplex DNA oligo fragment. - In the evaluation of the CRISPR-edited CGIs, the methylome observation at CpG locations within the tar- geted CGIs were bisulfite sequenced at levels above that of the overall respective genome-wide CpG bisulfite se- quences (Fig. - These observations increase the confi- dence in the statistical evaluations for the CpGs of the edited CGIs.. - In both edits, a marked increase of methylation is observed within the affected CGIs for the CpG loca- tions spanning the range of genomic incorporation of the donor DNA. - In the mechanics of the edit, the NHEJ edit did not replace the genomic sequence of the CGI with donor DNA recombination template dur- ing its application, leaving the endogenous methylation pattern unmodified. - 6 are not the result of CpG-free DNA integration and map closely to the homology arms of the donor DNA. - It has been reported that using CRISPR to modify the methylome patterns of the cell has impact on cellular function [33, 39]. - Disruptions of these methylation patterns within specific regions of the genome have been associated with disease states such as cancer and neurological disorders [43]. - For example, in the case of imprinting, methylation of a given allele of a gene from either the maternal or paternal strand can si- lence the given allele for the lifetime of the cell [44].. - From their results, they concluded that the methylation of elements in the promoter region of a gene can functionally silence the expression of the gene.. - Further, the modifica- tion of the methylome pattern represents a genomic scar caused by the application of a directed CRISPR cut and subsequent incorporation of donor DNA into the gen- ome. - Wiles of The Jackson Laboratory , Bar Harbor, ME, USA. - For replicate animals for each strain, genomic DNA (gDNA) was extracted from excised spleen tissues of the respective strains and processed for overall genomic methylation patterns. - gDNA was isolated using a modified protocol of the Genomic DNA Isolation Kit (AbCam. - gDNA was isolated from the extraction mixture using a modified protocol of the paramagnetic bead DNA isolation in the ChargeS- witch gDNA Micro Tissue Kit (Thermo Fisher Scientific;. - Bisulfite conversion of the fragments was performed using the EZ DNA Methyla- tion—Lightning kit (Zymo Research. - The size and concentration of the fragments were confirmed using an Agilent 2200 TapeStation. - alignment map (SAM) files were sorted using SAMtools sort of the SAMtools suite version 1.7 [51], converting the SAM files to sorted binary sequence/alignment map (BAM) files. - The first step evaluates the methylome data for genome-wide CGIs in a statistical hypothesis test of methylation level differences between the CGIs of the experimental and control animals. - Where μ d is the mean methylation level difference of the genetically-edited animal methylation level from the control animal methylation level at each CpG site within the defined CGI range. - A second filter was then applied to the paired t -test re- sults to filter biological epigenetic noise inherent to the epigenetic buffering of the transcriptome [56]. - In apply- ing this filter, CpGs within the CGI ranges were consid- ered only where the genetically-edited animal CpG methylation was greater than the CpG methylation of the control animal. - The remaining positive CpG methy- lation differences, resulting from the subtraction of the qualifying CpG methylation percentage of Control 1 Animal from those of CRISPR-edited animals or the Control 2 Animal, were then binned into the following. - Methylation profiles from the genomic regions of the animals within the alignment BedGraph files generated by analysis in MethylDackel were mapped to the Mus musculus (house mouse) refer- ence genome version GRCm38 (mm10).. - In each comparison and at each qualifying CpG location, the percent methylation values observed for the Control 1 Animal were subtracted from the qualifying CpG locations of the CRISPR-edited ani- mal and the Control 2 Animal. - Results of the analysis provided a basis for determining targeted ranges for evaluating clustered CpG methylation variance across genomes.. - Difference profiles of the NHEJ1 Animal compared to Control 1 Animal of the two most significantly changed CGIs — Chr A) and Chr B. - 5 and 6 display uniform increase in methylation in the CpGs of the CGI above that of the Control. - MHF, PAT, AAM, MVW, EFMG, SAK, and KR provided analyses and assisted in the development of the manuscript. - This study was conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of The National Academies. - CRISPR/Cas9-based engineering of the Epigenome. - The genome of C57BL/6J "eve", the mother of the laboratory mouse genome reference strain. - Picard MarkDuplicates Broad Institute of the Massachusetts Institute of Technology and Harvard University. - Proceedings of the 9th Python in Science Conference
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