In VivoBlunt-End Cloning Through CRISPR/Cas9-Facilitated Non-Homologous End-Joining

Abstract

The ability to precisely modify the genome in a site-specific manner is extremely useful.The CRISPR/Cas9 system facilitates precise modifications by generating RNA-guided doublestrand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called knock-in blunt ligation for exploiting this excision and repair to insert exogenous sequences in a homology-independent manner without loss of additional nucleotides. We successfully utilize this method in a human immortalized cell line and induced pluripotent stem cells to insert fluorescent protein cassettes into various loci, with efficiencies up to 35.8% in HEK293 cells. We also present a version of Cas9 fused to the FKBP12-L106P destabilization domain for investigating repair dynamics of Cas9-induced double-strand breaks. Our in vivo blunt-end cloning method and destabilization-domain-fused Cas9 variant increase the repertoire of precision genome engineering approaches.

CRISPR/Cas9, a universal tool for genomic engineering

“…Это может приводить к по тере или вставке нуклеотидов (индел-мутации), а также к нуклеотидным заменам. Частота Cas9-обусловленного мутагенеза обычно составляет несколько десятков про центов (Geisinger et al, 2016).…”

CRISPR/Cas9-mediated traceless gene correction and activation of the HBB locus in iPSCs with the beta thalassaemia mutation

“…Translocation through precise end joining was demonstrated for blunt end DSBs induced by Cas9WT [121]. Moreover, a recent study demonstrated a high percentage of precise end joining between two blunt end DSBs that generated genomic microdeletions in different cell types [120], lending further support to the idea that blunt ends generated by Cas9WT can be repaired through error-free NHEJ. It should be noted, however, that in the study by Geisinger et al [120] deletion of the intervening sequence would have prevented the iterative cycle of cleavage and repair induced by the nuclease [114].…”

“…Moreover, a recent study demonstrated a high percentage of precise end joining between two blunt end DSBs that generated genomic microdeletions in different cell types [120], lending further support to the idea that blunt ends generated by Cas9WT can be repaired through error-free NHEJ. It should be noted, however, that in the study by Geisinger et al [120] deletion of the intervening sequence would have prevented the iterative cycle of cleavage and repair induced by the nuclease [114]. Upon further examination of the data in that report, the range of precise ligation was higher in H9 pluripotent stem cells (>75 per cent, except for one guide RNA) than in HEK 293 cells (which varied between 44 per cent and 90 per cent), despite a similar repair pattern in both cell types.…”

“…The DNA termini (3' or 5') generated by Cas9 are not damaged and therefore may facilitate the restoration of the sequence by religation (i.e. C-NHEJ or BER) without end resection [97,120]. Translocation through precise end joining was demonstrated for blunt end DSBs induced by Cas9WT [121].…”

“…Furthermore, As has already been discussed in the previous paragraph, NHEJ can result in an errorfree DNA repair [120].…”