Suppression of NFIb expression by gene targeting strategies

Transcription factors play an important role in the development of the central nervous system and regulation of stem cell function. One example of such transcription factors is the Nuclear Factor I (NFI) family which consists of four members in vertebrates: NFIA, NFIB, NFIC and NFIX. Of the three NFI family members, NFIA, NFIB and NFIX are expressed in the in the developing nervous system and the adult brain in an overlapping but distinct pattern, leading to similar brain developmental defects when deleted. NFI proteins carry a conserved DNA-binding domain at the amino terminus and a CTF-1 transcriptional modulatory region at the carboxy terminus. Through the N-terminal and C-terminal regions NFI proteins can interact with each other and other proteins respectively. NFI proteins recognize and bind to the TTGGC(N5)GCCAA consensus sequence on genomic DNA as heterodimers or homodimers. NFI proteins have been shown to either activate or inhibit gene expression, depending on the specific promoter and cellular and developmental context. In mammals, the presence of four NFI genes with possibly overlapping functions makes it difficult to determine the in vivo targets of individual NFI proteins and the specific roles of NFI members in development. In the central nervous system, NFI family plays an important role in controlling a number of important processes such as axonal guidance and development, glial and neuronal cell differentiation as well as neuronal cell migration. The presence of NFIs is required for proper axonal growth and dendrite formation in cerebellar granule neurons in vitro. In the hindbrain, Nfia and Nfib are highly expressed in the spinal cord at the beginning of gliogenesis. Overexpression or knockdown of Nfia and Nfib in the chick embryo has shown that both are necessary for spefication of glial fates in the developing spinal cord. Elsewhere in the hindbrain, Nfib regulates the migration and terminal differentiation of cerebellar granule neurons. In the forebrain, Nfib is expressed in the proliferative region and in the subcerebral and corticothalamic projection neurons, and regulates neuronal progenitor maintenance and differentiation. Thus, NFI proteins and NFIB in particular are important regulators of neural development and their functions have been widely studied but have not yet been fully elucidated via functional experiments in mice and chick. However, the individual functions of these genes have not yet been discerned. One recent technology to carry out loss of function studies is the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system. While recombinant DNA technology developed in the 1970s, allows manipulation of DNA, advance of nuclease-based genomic editing technologies was required to target the very large eukaryotic genomes. These technologies generally take advantage of two double strand break repair mechanisms: Nonhomologous end-joining (NHEJ) and Homology-directed repair (HDR). NHEJ can lead to errors during the ligation of the broken DNA molecule, which can lead to the insertion or deletion of short base pairs in the region where the DNA is cleaved. HDR can be used to specifically insert or replace targeted DNA. Zinc finger and TALE (transcription activator-like effector) proteins can recognize specific sequences on DNA. Recombinant nucleases (ZFN and TALENs), which are the result of fusing these proteins with FokI nuclease, are used to cleave specific genomic locations and create double strand breaks. However, this approach has limitations: as Zinc Finger domains are expanded to target longer sequences, the DNA binding specificity can be altered. Similarly, the DNA binding specificities of the TALENs can be modified by addition of TALE modules, however, generation of new recombinant TALENs is costly and complex. The alternative to these methods is the CRISPR/Cas9 system, which is based on microbial adaptive immune system. In the CRISPR/Cas9 systems, the Cas9 enzyme forms double strand breaks in the target region. A short guide RNA (sgRNA) can target almost any region on the genome. CRISPR systems includes the Cas9 nuclease, the crRNA that express guide RNAs and trans-activating crRNA (tracrRNA) that facilitates processing of the crRNA array to de different units. Each crRNA unit includes a 20 nucleotide guide sequence and a partial direct repeat, that the former directs Cas9 to a 20 base pairs DNA target through Watson-Crick base pairing. For genome editing, crRNA and tracrRNA fused into a single guide RNA (sgRNA). Cas9 can be targeted via changing the 20 nucleotide guide sequence adjacent to the PAM sequence. The sgRNA will be provide the scaffold for Cas9 binding to DNA. The sequence of sgRNA can be modified to target to region of interest on the genome. sgRNAs are generally around 100 nucleotides long. From the 5' end, 20 nucleotides anneal to desired DNA sequences via Watson-Crick base pairing and direct the Cas9 to cleave the desired genomic DNA. The rest of the double-strand structure at 3' is important for Cas9 identification. The sgRNA can inserted into cells as RNA or via a sgRNA expression vector. Crispr/Cas9 is used broadly in biological applications due to the simplicity of Cas9 targeting, high efficiency as a site specific nuclease and possibility of complex modifications. Nevertheless, there are also some limitations to CRISPR applications. Cas9 is able to recognize and cleave target DNA through guide sequence on the single guide RNA. Cas9 requires PAM sequences and but this requirement does not limit the range of Cas9 targets. However, Cas9 can also cleave off-target sites and generate off-target modifications The distincts functions of NFI proteins, and specifically NFIB, in regulation of gene expression in stem cells as well as in cancer remains to be elucidated. To carry out loss of function studies in human cell lines in vitro, we set out to knock-out and knock-down NFIB. We targeted the NFIB exon2 with CRISPR/Cas9 sgRNA constructs, set up a single cell isolation experiment and isolated 5 and 15 clones for sgRNA#1 and #2, screened 3 and 9 clones by T7 endonuclease assay respectively. However, no mutants were identified in this assay. We also generated shRNA constructs to target NFIB, and compared their efficacy with other shRNAs that target the NFI family members. Preliminary experiments show that a mixture of shNFIB constructs can decrease NFIB transcript levels by 60% in U-87 MG glioblastoma cells. Both approaches will be used to further studies of NFI function in human neural cell lines.