Development and application of transposable element technology

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Development and application of transposable element technology The human genome contains over 500,000 copies of L1, a non-LTR retrotransposon that accounts for 17% of the genome. A small number of these L1s are transposition competent and are responsible for the majority of de novo insertions including those causing over 124 cases of spontaneous genetic disorders. The majority of genomic L1s (>95 %) are grossly truncated at their 5' end. Full-length copies of L1 are 6 kb and encode its RNA Pol II promoter, an RNA binding protein called ORF1, and a combination endonuclease and reverse transcriptase designated ORF2. DNA vectors that express engineered L1s in cultured cells reveal many features of L1 transposition including the roles of ORF2 in making a nick at insertion sites and in reverse transcription of the 1st strand using the nick as primer. Painstaking analysis of individual de novo insertions confirmed the high frequency of 5' truncation, which renders the elements nonfunctional for transposition; however, the mechanism of the 5' truncations is unknown. The hypothesis that the RT activity of ORF2 has poor processivity is unlikely as in vitro assays demonstrate high processivity. What seems more likely is that 5' truncation is the outcome of genetic conflict with host factors that serve as defenders of the genome. We aim to identify the molecular mechanism of 5' truncation of newly transposed L1 elements. Engineered vectors expressing L1 containing antisense reporter genes such as GFP allow measurements of de novo integration in cultured cells (HeLa or HEK293T). The reporter genes are expressed only after transposition due to an antisense intron that is spliced before reverse transcription. Unfortunately, these reporter genes cannot be used to measure 5' truncation because they are placed in the 3' UTR to preserve L1 coding function. To detect 5' truncations, we designed an antisense reporter gene in the 5' UTR of L1 engineered vectors. There are many variables that could prevent these new L1 vectors from measuring transposition. A reporter gene in the 5' UTR could disrupt critical functions of L1 such as promoter activity, ORF1 translation, or reverse transcription. To identify active L1 vectors we chose a total of 12 designs with various positions in the 5' UTR and four versions of RFP and mCherry reporter genes. We have now identified a functional 5' UTR reporter that can determine overall levels of 5' truncation in cell lines by determining what fraction of cells express both the GFP and the mCherry proteins. These are the cells with full length L1 integration. We have used this dual reporter system in HEK293T cells and have visualized the presence of both red and green fluorescence with microscopy. Dual reporter L1 with mutations in ORF1 and in ORF2 demonstrate that the florescence is due to de novo integration. Importantly, integration frequencies of dual reporter L1 as determined by GFP expression are equivalent to L1 lacking the 5' mCherry. This demonstrates that the reporter in the 5' UTR does not reduce integration activity. Comparing the ratios of red+green to green reveals full-length insertions represent approximately 7% of all integration. Previous studies identified two resides in L1 that appeared to alter the length of insertions. We found that mutations of these residues lowered the ratio of full-length to 5' truncated insertions by 2-fold. Experiments with dual reporter L1 focus on the identification of host factors that affect the percentage of full-length insertions. A large siRNA depletion screen identified a host factor that inhibits the frequency of full-length integration by approximately two-fold. In vitro assays of reverse transcription reveal that this factor reduces the length of the cDNA products. Current experiments are focused on testing whether this host factor interacts directly with L1 RNA during reverse transcription. A Modular Toolkit for Genome Editing in Schizosaccharomyces pombe Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR)-Cas is a bacterial defense system which has been widely adopted as a method for gene editing. The single-guide RNA (sgRNA), complementary to the target sequence, allows the Cas protein to bind and create double stranded breaks in DNA that when repaired can alter sequence. In addition, the dCas9, where the endonuclease domain of cas9 is mutated, can repress transcription by blocking RNA Polymerase II from transcribing through the sequence. However, a system of flexible vectors that express CRISPR components is not currently available for use in Schizosaccharomyces pombe. Here, we developed a CRISPR toolkit with unique capabilities that express Cas9 and multiplexed sgRNAs that delete entire genes or instead express inactive Cas9 (dCas9) that inhibits gene expression. Through Golden Gate Assembly and a standardized set of restriction site overhangs, we provide flexible modules that allow the user to readily assemble plasmids containing a choice of constitutive or inducible Cas9/dCas9, sgRNAs, multiplexed sgRNA, and selectable genes for maintaining the plasmids in S. pombe. We have validated the ability of our system to delete an entire gene by removing ade6. The absence of this gene results is red pigmented colonies, a phenotype that revealed approximately 50% of cells with the active Cas9 module lack ade6 activity. PCR amplification and sequencing of the locus from pigmented colonies revealed that most had the desired deletion of the entire ade6 gene. By replacing the active Cas9 with the dCas9 module we tested the ability of our system to inhibit gene expression. The colonies expressing dCas9 and sgRNAs effectively inhibited transcription of ade6 as indicated by red pigmented colonies. We have also tested our CRISPR modules on ura4. Deletion of ura4 or inhibition of its expression results in an observable phenotype, resistance to 5-fluoroorotic acid. This toolkit provides the flexibility of golden gate modules with multiple selectable markers, multiplexed sgRNAs, and inducible expression of Cas9. These features will provide the community of scientists working with S. pombe efficient new methods to alter gene expression.