Microhomologies are prevalent at Cas9-induced larger deletions

The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone.

Characterization and Expression of an In-Frame Exon 33-34 Deletion Causing Type 1 VWD

Introduction: In recent years, the role of copy number variation (CNV) in the pathogenesis of type 1 von Willebrand disease (VWD) has been extensively explored. Large cohort studies including the MCMDM-1VWD and French studies have identified a total of eight partial VWF gene deletions, involving single and multiple exons, as well as one duplication. Furthermore, breakpoints have been identified within the MCMDM-1VWD cohort study for deletions involving exons 3, 32-34 and 33-34. Aim: To characterize and determine heterozygous deletion breakpoints of a deletion within the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD) cohort, previously identified through array CGH analysis and to determine the pathogenesis of the deletion through in vitro analysis. Methods: Identification of deletion breakpoints utilized a deletion-specific multiplex PCR approach, whereby two forward primers and one reverse primer were designed to determine the heterozygous exon deletion, based on previous optimization within the MCMDM-1VWD cohort. VWF antigen (VWF:Ag), VWF propeptide (VWFpp), and multimer distribution were performed in a central laboratory. Recombinant exon 33-34 deletion mutant (rVWFdel33-34) was created using site directed mutagenesis. Recombinant vectors were transiently transfected into HEK293T cells and Renilla used as a transfection control. Conditioned media and cellular lysates were collected 48hr post-transfection and analysed by VWF:Ag ELISA, comparing to wild-type VWF (rVWFwt) expression. Results: Three families enrolled in the ZPMCB-VWD displayed a deletion of VWF exon 33-34. The deletion was determined to be 3.4kb in size and led to an in-frame loss of exons 33-34, with breakpoints being identical to those identified in the MCMDM-1VWD study (c.5620+872_5842+2440delinsGCAGCATAAGCATAAAGC). VWFpp/VWF:Ag ratios were elevated in all affected family members with the exon 33-34 deletion (mean 4.3) as opposed to unaffected family members (mean 1.3). These results are consistent with findings in the MCMDM-1VWD family (mean VWFpp/VWF:Ag of 4.8), suggesting increased clearance of VWF. In silico analysis of the breakpoint region suggested no significant repetitive elements and microhomology mediated end-joining was hypothesised as the potential mechanism leading to the deletion. Expression of recombinant mutant rVWFdel33-34 revealed that secretion reduced by 52% in the homozygous state (p<0.0001) when compared to rVWFwt expression. The deletion also resulted in a significant 35% reduction in secretion in the heterozygous state (p<0.0001). However, no intracellular retention was observed in either mutant state. Conclusion: Analysis of the exon 33-34 deletion breakpoint revealed no highly homologous repetitive sequences but there was a DNA insertion of 18 bp at the deletion breakpoint. In vitro expression of the exon 33-34 deletion suggests that there is a moderate effect on VWF secretion into conditioned media, without increased intracellular retention. The exon 33-34 deletion leads to the loss of nine cysteine residues. Inter- and intra- disulphide bonding between cysteine residues within VWF monomers and multimers is known to be functionally important in facilitating correct VWF structural orientation and folding within D4-CK domains. Loss of these cysteines may explain the moderate reduction in VWF secretion and potentially reduced plasma survival of the variant VWF. Disclosures Montgomery: Immucor: Patents & Royalties.

The albino-deletion complex of the mouse: molecular mapping of deletion breakpoints that define regions necessary for development of the embryonic and extraembryonic ectoderm.

Previous complementation analyses with five (c11DSD, c5FR60Hg, c2YPSj, c4FR60Hd, c6H) of the mouse albino deletions defined at least two genes on chromosome 7, known as eed and exed, which are necessary for development of the embryonic and extraembryonic ectoderm, respectively, of early postimplantation embryos. The region of chromosome 7 containing these two genes has now been accessed at the molecular level by cloning two of the deletion breakpoint-fusion fragments. The c2YPSj breakpoints were isolated by cloning an EcoRI fragment containing a copy of an albino region-specific repeat unique to c2YPSj DNA. Similarly, the c11DSD breakpoints were isolated by cloning a c11DSD EcoRI fragment detected by a unique-sequence probe mapping proximal to the albino-coat-color locus. By mapping the cloned breakpoints relative to the remaining three deletions, the c11DSD distal breakpoint was found to define the distal limit of the region containing eed, whereas the c2YPSj and c6H distal breakpoints were found to define the proximal and distal limits, respectively, of the region containing exed.