HR-based genome editing requires that both a nuclease and a donor DNA molecule be provided to the cell. In HR-based editing, the cell’s natural HR machinery uses the engineered undamaged donor as the template for the repair of the nuclease-induced break. To differentiate HR that repairs a DSB using the sister chromatid as a natural form of repair from genome editing HR, the term “homology-directed repair” (HDR) has been coined to describe the application for genome editing (Fig.1). In nuclease-based gene targeting, the donor vector needs to have flanking homology arms of greater than 400 bp (although shorter can work).[1] These relatively short homology arms are in contrast to the several thousand base pair homology arms needed for mouse ES cell gene targeting.

Fig1. VARIATIONS ON EDITING USING HOMOLOGY-DIRECTED REPAIR/HOMOLOGOUS RECOMBINATION.

There are a wide variety of changes that can be made using HDR based genome editing that can be used for both research and therapeutic purposes. These include:

1. Making single nucleotide changes to the genome, such as to revert a disease-causing genetic single-nucleotide variant;

 2. Inserting a short stretch of nucleotides at specific location, such as to add an epitope tag to a protein;

3. Inserting a full complementary DNA (cDNA) or partial super exon cDNA back into a gene to functionally correct downstream mutations;

4. Inserting one gene into another gene or replacing one gene with another gene. In this form of synthetic biology, the genome is being reprogramed to express the inserted gene under the control of the target gene’s regulatory apparatus.

 5. Inserting one gene into another gene without disrupting the target gene. Thus the knockin gene is now expressed along with the target gene in the cell.

 6. Inserting promoter-gene cassette(s) into a safe harbor locus. A safe harbor is defined as a genomic location that if DNA is inserted into it, there would be no deleterious consequence. A safe harbor can be universal, in which it would safe to insert in all cell types or can be cell type specific, where insertion is safe in that cell type but not in another. There are many possible safe harbor options, but each needs to be carefully evaluated.

A version of HDR is to use a single-stranded oligonucleotide (ssODN) as a template rather than a gene targeting donor vector. The mechanism by which ssODNs are used by the cell to repair a DSB is not through the standard recombination pathway but instead through a pathway called “single-stranded template repair” (SSTR). SSTR does not require canonical HR genes such as Rad51 but instead requires genes in the Fanconi anemia family.[2,3] The nonnuclease targeted integration of single-stranded AAV vectors also requires genes from the Fanconi anemia family.[4] Presumably SSTR-based editing, like HR based editing, is harnessing a natural pathway that cells use to repair DSBs, but the natural function of SSTR, unlike HR, has not been well established.

References

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[1] Hendel A, et al. Quantifying on- and off-target genome editing. Trends Biotechnol. 2015;33(2):132–140.

[2] Gallagher DN, et al. A Rad51-independent pathway promotes single-strand template repair in gene editing. PLoS Genet. 2020;16(10):e1008689.

[3] Richardson CD, et al. CRISPR-Cas9 genome editing in human cells occurs via the Fanconi anemia pathway. Nat Genet. 2018;50(8):1132–1139.

[4] de Alencastro G, et al. Improved Genome Editing through Inhibition of FANCM and Members of the BTR Dissolvase Complex. Mol Ther. 2021;29(3):1016–1027.

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مواضيع ذات صلة

Genotoxicity Through Potential Off-target Nuclease - Induced Double - Strand Breaks

Early Transcription of Genes N and Cro

Lambda’s Relatives and Lambda Hybrids

The Genetic Structure of Lambda

The Physical Structure of Lambda

Other Attenuated Systems: Operons, Bacillus subtilis and HIV

Coupling Translation to Termination

trp Multiple Secondary Structures in trp Leader RNA

Early Explorations of the Hypersynthesis

The Serendipitous Discovery of trp Enzyme Hypersynthesis

Rapid Induction Capabilities of the trp Operon

The Aromatic Amino Acid Synthetic Pathway and its Regulation