Large genomic fragment deletions and insertions in mouse using CRISPR/Cas9

Abstract

ZFN, TALENs and CRISPR/Cas9 system have been used to generate point mutations and large fragment deletions and insertions in genomic modifications. CRISPR/Cas9 system is the most flexible and fast developing technology that has been extensively used to make mutations in all kinds of organisms. However, the most mutations reported up to date are small insertions and deletions. In this report, CRISPR/Cas9 system was used to make large DNA fragment deletions and insertions, including entire Dip2a gene deletion, about 65kb in size, and β-galactosidase (lacZ) reporter gene insertion of larger than 5kb in mouse. About 11.8% (11/93) are positive for 65kb deletion from transfected and diluted ES clones. High targeting efficiencies in ES cells were also achieved with G418 selection, 46.2% (12/26) and 73.1% (19/26) for left and right arms respectively. Targeted large fragment deletion efficiency is about 21.4% of live pups or 6.0% of injected embryos. Targeted insertion of lacZ reporter with NEO cassette showed 27.1% (13/48) of targeting rate by ES cell transfection and 11.1% (2/18) by direct zygote injection. The procedures have bypassed in vitro transcription by directly co-injection of zygotes or co-transfection of embryonic stem cells with circular plasmid DNA. The methods are technically easy, time saving, and cost effective in generating mouse models and will certainly facilitate gene function studies.

Fig 1. Circular plasmid mediated large genomic deletion in mES cells.

(A) Dip2a gene genomic locus, sgRNAs cutting sites and genotyping strategy. Blue arrows indicate genotyping primers. (B) Optimization of mouse embryonic stem cell nucleofection with pCBh-EGFP-N1 plasmid. (C) Genotyping nucleofected mES pool for ∼65kb deletion.

Fig 2. Plasmid mediated long range homologous recombination in mES cells.

(A) Targeting strategy of Dip2a gene. The designed primer pairs for screening are indicated with blue and green. (B) Schematic illustration of nucleofection and selection. The targeted clones in red and non-targeted in pink. (C) Recombination screening of left and right arm by PCR. (D) Targeting efficiency with sgRNAs and DONOR in mES cells.

Fig 3. Circular plasmid mediated high efficiency deletion in zygotes.

(A) Injection of two sgRNA-pX330 plasmids at 5ng/μl each into zygote. (B) Genotyping 14 pups with the same primers in Fig. 1. (C) PCR sequencing of 3 pups with deletion. The PAM sequence is highlighted in green, the targeting sites are in red and the deleted regions are in blue.

Fig 4. CRISPR-Cas9 mediated lacZ-NEO knockin.

(A) Overview of targeting strategy. The LacZ gene with NEO selection cassette is inserted ahead of Dip2a ATG start codon. (B) The non-knockin alleles were PCR amplified and sequenced. CRISPR/Cas9 mediate indels are shown in detail. (C) Targeting frequency of G418 resistant mES clones. (D) Genotyping of 18 pups from direct zygotes injection with sgRNA-pX330 and DONOR plasmids. Two are positive for LacZ PCR. (E) LacZ staining of E10.5 Dip2a ^LacZ/+ embryos. DIP2A is expressed moderately in the brain (arrowhead) and at high level in the spinal cord, dorsal root ganglion and trigeminal ganglion (arrow).