Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes

Abstract

The CRISPR/Cas system efficiently introduces double strand breaks (DSBs) at a genomic locus specified by a single guide RNA (sgRNA). The DSBs are subsequently repaired through non-homologous end joining (NHEJ) or homologous recombination (HR). Here, we demonstrate that DSBs introduced into mouse zygotes by the CRISPR/Cas system are repaired by the capture of DNA sequences deriving from retrotransposons, genomic DNA, mRNA and sgRNA. Among 93 mice analysed, 57 carried mutant alleles and 22 of them had long de novo insertion(s) at DSB-introduced sites; two were spliced mRNAs of Pcnt and Inadl without introns, indicating the involvement of reverse transcription (RT). Fifteen alleles included retrotransposons, mRNAs, and other sequences without evidence of RT. Two others were sgRNAs with one containing T7 promoter-derived sequence suggestive of a PCR product as its origin. In conclusion, RT-product-mediated DSB repair (RMDR) and non-RMDR repair were identified in the mouse zygote. We also confirmed that both RMDR and non-RMDR take place in CRISPR/Cas transfected NIH-3T3 cells. Finally, as two de novo MuERV-L insertions in C57BL/6 mice were shown to have characteristic features of RMDR in natural conditions, we hypothesize that RMDR contributes to the emergence of novel DNA sequences in the course of evolution.

CRISPR/Cas9 interrogation of the mouse Pcdhg gene cluster reveals a crucial isoform-specific role for Pcdhgc4

“…Reducing diversity in the Pcdhg gene cluster Furthermore, linked reads revealed an insertion including a transposable element and coding sequence from Anp32a which aligns to exons 4-7 of transcript Anp32a-201 without the intervening introns ( Fig 2B, S1D Fig). This phenomenon of the insertion of a transposable element along with coding sequence from an early expressed gene has been previously described in CRISPR genome editing [54]. As this sequence was inserted 3' to the inverted exon Pcdhga5, there is no associated transcription start site, and no protein product is expected.…”

Discovery and characterization ofAlurepeat sequences via precise local read assembly

“…Multiple non-TPRT mechanisms may have resulted in the duplication of these sequences. For example, they may have served as donor sequences utilized in replication template switching (87,88) or in templated double-strand break repair (89) as has been reported for CRISPR/Cas induced lesions (90). Several insertions had sequence characteristics indicative of subsequent deletion in the genome reference sequence, such as the presence of regions of overlap of unusual length (i.e., 21 bp overlap for the AluSx1 located at chr2:161952317, 98 bp for the AluSx3 at chr11:35425492), additional flanking non-Alu sequence that does not map to the human reference at that location (refer to alignments for sites chr1:232869263, chr6:5348761, and chr12:26958660 in Figure S6), and/or coincident presence with bimorphic insertions reported in the genomes of Great Apes and other primates (67) ( Table S5).…”

Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes

“…The frequent presence of sgRNA template sequence was particularly striking, as the N-oligo was approximately 1000-fold more abundant in the nucleofection reactions ( Extended Data Figure 5 ). The occasional insertion of non homologous DNA into double strand breaks has been reported in yeast 12-14 and mice 15-17 and the insertion of short phosphorothioate-protected oligos forms the basis of the GUIDE-Seq method to detect off-target genome editing events 18 , but the ability of non-homologous single stranded DNA to greatly increase gene knockout by stimulating these events is surprising and to the best of our knowledge unprecedented.…”