(Д«) A schematic diagram of the target sites in the chicken GAPDH 3вЂ™UTR flanked with homology arms. Figure 1.ДЄ) A schematic illustration of the chicken GAPDH locus, HDR-cassette, edited GAPDH locus. The left homology arm was amplified by PCR with the following primers: 5вЂ™-TTGTTGACCTGACCTGCCGTCTGGAG-3вЂ™ and 5вЂ™-CTCCTTGGATGCCATGTGGACCATCAAG-3вЂ™ the right homology arm was amplified with primers 5вЂ™-CCCTTTGTTGGAGCCCCTGCTCTTC-3вЂ™ and 5вЂ™-GAGCCCTGTATCTTCCTTGCACAGACC-3вЂ™. Diphteria Toxin Fragment A (DTA) coding sequence was inserted in the shuttle after the right arm under a PGK promoter for negative selection (ДЇigure 1A). The total length of the inserted sequence between two homology arms in the shuttle was 3259bp. Neomycin resistance (neo) gene was cloned after eGFP under a constitutive cytomegalovirus (CMV) promoter for positive selection of cells with the desired insertion. Coding sequence of the left arm was in frame with P2A-eGFP in order to provide the gene transcription under the control of endogenous promoter. Left and right homology regions in the shuttle flank the P2A-eGFP sequence, where eGFP is enhanced green fluorescent protein.

Homology regions of 999bp and 3093bp for left and right arms respectively were amplified from the genomic DNA of chicken cell line DF1 by PCR, and cloned using MauBI and PmeI restriction sites for the left arm, and SgrDI and AscI for the right arm respectively (DF-1 genome is yet to be sequenced, common chicken genomic data is available The targeting vector was designed based on the plasmid LSL-Cas9-Rosa26TV (#61408, Addgene). A plasmid pQE30TaqRFP (Evrogen Moscow, Russia) coding RFP was used for cotransfection as a reporter of the efficiency of DNA delivery to the cells. Unique 20bp sequences for the selected gRNAs were cloned under human U6 promoter in the plasmid phU6-gRNA (#53188, Addgene). Similarly as for Cas9 it has been previously demonstrated that the human U6 promoter works in chicken cells. A plasmid CAG-Cas9 (#89995, Addgene Cambridge MA, USA) was taken for human codon-optimized Cas9 expression. We used human codon-optimized Cas9 (hCas9) as it has been previously demonstrated that the optimized Cas9 works in chicken cells and there was no need to synthesize chicken codon-optimized nuclease. Methods Construction of expression vectors

The result indicates that this system can be used as a tool for chicken genome editing. Here, we show the system to precisely insert a gene under the control of an endogenous promoter and select the cells with the successful integration. However, the ability not only to knock-out genes, but also to induce a tissue-specific expression of a gene of interest without destroying its endogenous locus would be beneficial. The CRISPR/Cas9 tool is a novel instrument compared with TALEN and some aspects of successful targeting with the Cas9 nuclease still need to be elucidated for avian species. Meanwhile CRISPR/Cas9 was used only to knock out genes in poultry (Д®t al., 2016]. There are some examples of successfully generated genetically modified chickens with usage of TALEN nuclease (Transcription Activator-Like Effector Nuclease). Nuclease-mediated gene insertion is several orders of magnitude more efficient compared with spontaneous recombination of DNA template alone that makes CRISPR/Cas9 an effective tool for genome editing.ДЄlthough CRISPR/Cas9-mediated gene editing has been widely used in a lot of organisms, this tool still has been rarely applied in avian species. The double strand break can be repaired in an error-prone way by non-homologous end joining (NHEJ), leading to small insertions/deletions, or by homology-directed repair (HDR), when a donor DNA-template is added [ Cas9 cuts double stranded DNA at the site specified by the guide RNA (gRNA). Clustered regularly interspaced short palindromic repeat (CRISPR), and CRISPR-associated nuclease (Cas9) - CRISPR/Cas9 are now widely used as an efficient method for genetic modification in a wide variety of organisms. However, retroviral delivery of an inserted sequence is an ineffective method due to random distribution of integration sites, and has adverse effects. Traditionally, transgenic chickens were generated using retroviruses. Precise and effective genome editing is one of the most important aspects in creating genetically modified organisms. Genetically modified chickens have great potential in agriculture, industry, biological research and pharmaceuticals.

Chimera tool rm 917