Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos

Abstract

11Introducing useful traits into livestock breeding programs through gene knock-ins has 12 proven challenging. Typically, targeted insertions have been performed in cell lines, followed by 13 somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce 14 genome editing reagents and a homologous recombination (HR) donor template into embryos to 15 trigger homology-directed repair (HDR). However, the HR pathway is primarily restricted to 16 actively dividing cells (S/G2-phase) and its efficiency is low in zygotes, especially for the 17 introduction of large DNA sequences. The homology-mediated end joining (HMEJ)-based 18 strategy harnesses HDR by direct injection of embryos, and has been shown to have an improved 19 knock-in efficiency in non-dividing cells. The knock-in efficiency for a 1.8kb gene was contrasted 20 when combining a gRNA/Cas9 ribonucleoprotein complex with either a traditional HR donor 21 template, or a HMEJ template in bovine zygotes. The HMEJ template resulted in a significantly 22 higher rate of gene knock-in as compared to the HR template (37.0% and 13.8%; P < 0.05). 23Additionally, more than a third of the knock-in embryos (36.9%) were non-mosaic. This approach 24 will facilitate the one-step introduction of gene constructs at a specific location of the bovine 25 genome and contribute to the next generation of elite cattle. 26 27Many attempts have been made to increase the rate of homologous recombination (HR) or 47 decrease the rate of non-homologous end joining (NHEJ) for gene insertion when using the 48 CRISPR/Cas9 system via CPI of zygotes 11 . However, these approaches have been unsuccessful in 49 bovine embryos as HR is primarily restricted to actively dividing cells 12 . Alternative homology 50 directed repair (HDR) approaches have been utilized for KI using a donor template via the 51 homology-mediated end joining (HMEJ) method 13 . This method has been shown to be active in 52 gametes and early stage 1-cell embryos, in which proteins necessary for pushing DNA repair 53 machinery towards the end-joining pathways are at their highest concentration 11 . While NHEJ 54 utilizes the Ku proteins and a ligase to mend the double-strand break (DSB) by blunt end ligation, 55 the HMEJ approach utilizes proteins for resection of the 5' ends and annealing of homologous 56 regions between the DSB and donor template similar to that in the microhomology-mediated end 57 joining (MMEJ) pathway 14 . 58In this study, we employed the HMEJ method for the precise insertion of the sex-59 determining region Y (SRY) gene into a region 10kb downstream of the zinc finger, X-linked (ZFX) 60 gene on the X chromosome of bovine embryos by injecting either in vitro matured oocytes prior 61 to in vitro fertilization, or presumptive zygotes 6hpi. We used two donor templates to compare KI 62 efficiency using the HMEJ and HR approaches, and show an increased rate of gene insertion at 63 the target location when using the HMEJ donor template. 64 RESULTS 65 Guide-RNA design and testing 66...

One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes

“…Collectively, these data suggest that a biallelic edit at the zygote stage, one of which was repaired by NHEJ resulting in a 26 bp insertion, and the other 38 kb complex allele knock-in which appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, and rearrangement following editing of the gRNA target site in the donor template. Multiple copies of the complete template in both forward and reverse orientations in the 38 kb allele was not expected with the HMEJ-mediated strategy, and was not observed in our previous study using this approach [10]. Such concatenation is more typical of homology independent targeted insertion (HITI) [25].…”

“…Although we achieved a 40% knock-in rate as determined by GFP expression in blastocysts, only 22 (11%) of the~200 microinjected embryos developed to the blastocyst stage. We previously observed a significant reduction in blastocyst development following microinjection of editing regents into MII oocytes (10.2%) and presumptive zygotes 6 hpi (17.6%), as compared to non-injected controls (29.3%) [10,15]. Additionally, only one of the nine embryos transferred resulted in a live birth.…”

“…In this experiment we chose to use a fluorescent marker to identify blastocysts with the SRY knock-in, and to target an autosomal safe harbor locus following our previous unsuccessful attempts to obtain live calves when targeting the SRY to a X chromosome locus and screening for knock-ins using embryo biopsy [10]. It is possible that the inclusion of the SV40 promoter to drive the expression of the GFP gene could result in the silencing of the adjacent SRY gene, as has been commonly observed with the hypermethylation of the cytomegalovirus (CMV) promoter.…”

“…Multiple repair pathways are thought to be involved in mediating a gene knock-in using this method. Previously, we found that the use of a HMEJ repair template to target an insertion to the X chromosome increased the knock-in frequency in bovine embryos as compared to a traditional HR template [9], and that more than a third of knock-in blastocysts analyzed were non-mosaic with precise integrations [10]. Blunt end ligation of cleaved donor template by homology independent insertion was also observed, more frequently in male than female embryos, but no integration of the donor plasmid backbone was ever detected [10].…”