Histone Modifications of H3K4me3, H3K9me3 and Lineage Gene Expressions in Chimeric Mouse Embryo.

OBJECTIVE: Chimeric animal exhibits less viability and more fetal and placental abnormalities than normal animal. This study was aimed to determine the impact of mouse embryonic stem cells (mESCs) injection into the mouse embryos on H3K9me3 and H3K4me3 and cell lineage gene expressions in chimeric blastocysts. MATERIALS AND METHODS: In our experiment, at the first step, incorporation of the GFP positive mESCs (GFP-mESCs) 129/Sv into the inner cell mass (ICM) of pre-compacted and compacted morula stage embryos was compared. At the second and third steps, H3K4me3 and H3K9me3 status as well as the expression of Oct4, Nanog, Tead4, and Cdx2 genes were determined in the following groups: i. In vitro blastocyst derived from In vivo morula subjected to mESCs injection (blast/chimeric), ii. In vivo derived blastocyst (blast/In vivo), iii. In vitro blastocyst derived from culture of morula In vivo (blast/morula), and iv. In vitro blastocyst derived from morula In vivo subjected to sham injection (blast/sham). RESULTS: Subzonal injection of GFP-mESCs at the pre-compacted embryos produced more chimeric blastocysts than compacted embryos (P<0.05). The number of trophectoderm (TE), ICM, ICM/TE and total cells in chimeric blastocysts were less than the corresponding numbers in blastocysts derived from other groups (P<0.05). In ICM and TE of chimeric blastocysts, the levels of H3K4me3 and H3K9me3 were respectively decreased and increased compared to the blastocysts of the other groups (P<0.05). Expressions of Oct4, Nanog and Tead4 were decreased in chimeric blastocysts compared to the blastocysts of the other groups (P<0.05), while this was not observed for Cdx2. CONCLUSION: In the present study, embryo compaction significantly reduced the rate of incorporation of injected mESCs into the ICM. Moreover, in chimeric blastocysts, the levels of H3K9me3 and H3K4me3 were altered. In addition, the expressions of pluripotency and cell fate genes were decreased compared to blastocysts of the other groups.

Efficient Production of Biallelic RAG1 Knockout Mouse Embryonic Stem Cell Using CRISPR/Cas9.

BACKGROUND: Recombination Activating Genes (RAG) mutated embryonic stem cells are (ES) cells which are unable to perform V (D) J recombination. These cells can be used for generation of immunodeficient mouse. Creating biallelic mutations by CRISPR/Cas9 genome editing has emerged as a powerful technique to generate site-specific mutations in different sequences. OBJECTIVES: The main purposes of this study were to achieve complete knock-out of RAG1 gene by investigating the nature of mutations in mutant mESC and to generate RAG1 knock-out mESCs containing homozygous indels with the aim of creating desired and specific RAG-1 -/- mutant mouse in a shorter period of time. MATERIALS AND METHODS: Here, we first utilized CRISPR/Cas9 system to target RAG1/RAG2 genes in NIH3T3 cells to test the activity and efficiency of our CRISPR system. Then we used the system for targeting RAG1 gene in mouse embryonic stem cell (mESCs) to generate knock-out embryonic stem cells. This method combined with highly active single guide RNA (sgRNA) is an efficient way to produce new RAG1-knockout mESCs in the selected regions of early coding DNA sequence, approximately between nucleotide c. 512-c. 513 and nucleotide c. 725-c. 726 of RAG1 coding sequence that had not been targeted previously. RESULTS: CRISPR gene editing resulted in a multitude of engineered homozygous and compound heterozygous mutations, including both in-frame and out-of-frame indels in 92% of mES cell clones. Most of the mutations generated by CRISPR/Cas9 system were out-of-frame, resulting in a complete gene knockout. In addition, 59% of the mutant ES cell clones carried out-of-frame homozygous indel mutations. The RAG1-knockout mESC clones retained normal morphology and pluripotent gene expression. CONCLUSIONS: Our study demonstrated that CRISPR/Cas9 system can efficiently create biallelic indels containing both homozygous and compound heterozygous RAG1 mutations in about 92% of the mutant mESC clones. The 59% of mutant ES cell clones carried out-of-frame homozygous indel mutations.

In Vitro Pre-validation of Gene Editing by CRISPR/Cas9 Ribonucleoprotein.

BACKGROUND: The CRISPR/Cas9 genome editing system is a powerful and simple gene editing method. The format of the CRISPR components is one of the important factors in targeting efficiency. Compared to plasmid or mRNA (IVTs) format, using the CRISPR/Cas9 system as Cas9-crRNA-tracrRNA RNP format is more efficient and rapid, especially in minimizing some of the pitfalls of CRISPR-mediated gene editing. In addition to efficient in vivo applications of the CRISPR RNP format in a variety of cell types and organisms, another advantage of this approach is usability for in vitro applications in which the crRNAs in the tracrRNA-crRNA structure guides the Mg2+-dependent RNAdirected DNA endonuclease to introduce double-strand breaks at specific sites in DNA. METHODS: Here, Cas9-crRNA-tracrRNA RNP system was used to test the designed crRNAs for in vitro DNA cleavage by Cas9 protein in RAG1, RAG2 and IL2RG genes. RESULTS: The results of cleavage reveal theCas9-crRNA-tracrRNA RNP system is a rapid and efficient way to pre-validate the efficiency of CRISPR cleavage with crRNAs designed for RAG1, RAG2 and IL2RG genes. CONCLUSION: one step in vitro cleavage of DNA by CRISPR/Cas9 ribonucleoprotein complex can be used to pre-validate the functionality and relative efficiency of CRISPR system for targeting genes.

CRISPR/Cas9 System for Efficient Genome Editing and Targeting in the Mouse NIH/3T3 Cells.

BACKGROUND: The Clustered, Regularly Interspaced, Short Palindromic Repeats (CRIS-PR) and CRISPR-associated protein (Cas) system has been used as a powerful tool for genome engineering. In this study, the application of this system is reported for targeting Rag genes to produce mutant mouse NIH/3T3 cell line. The Rag1 and Rag2 genes are essential for generation of mature B and T lymphocytes. Disruption of Rag genes causes disease like Severe Combined Immunodeficiency syndrome (SCID). Here, the efficiency and specificity of CRISPR system were tested with highly active sgRNAs to generate novel mutations in the NIH/3T3 mouse cell line. METHODS: Four single guide RNAs were designed to target sequences in the coding region of the Rag1 and Rag2 genes. Four sgRNA-CAS9 plasmids were tested to target Rag1 and Rag2. RESULTS: Based on T7 endonuclease assay and sequencing analysis, the expression of sgRNAs targeting two sites in Rag1 resulted in deletion of the intervening DNA fragment. The expression of sgRNAs with Cas9 targeting two sites in Rag2 gene resulted in indel mutations at both sites. In this report, fragment deletion in Rag1 gene was detected in about 50% of transfected cells. CONCLUSION: Therefore, CRISPR/Cas9 system can be highly efficient and specific when gRNAs are designed rationally and provides a powerful approach for genetic engineering of cells and model animals.