The spontaneous frequency of a mutation occurring at a specific site in a nontransformed human somatic cell is on the order of 10⁻⁸ to 10⁻⁹. Thus the ability to create specific mutations at a specific site in a reproducible fashion at high frequency is not generally possible. Moreover, the frequency of spontaneous targeted integration (the insertion of a DNA fragment at a specific location in the genome [also called gene targeting]) is on the order of 10⁻⁶ (only one in a million cells will undergo this process).[1] The identification of cells having undergone gene targeting can be increased by several orders of magnitude by using positive and negative selection strategies.[2-4]

The frequencies of targeted mutation and targeted integration change dramatically if a DNA double-strand break (DSB) is created at the target site.[5-7] Using contemporary methods to create DSBs, the frequency of targeted mutations, even in primary human cells, can now exceed greater than 90% of alleles and the frequency of targeted integration can routinely exceed greater than 50% of alleles. Therefore a site-specific DSB increases the frequency of targeted genome sequence changes (genome editing) by 6 to 9 orders of magnitude and is a key foundational principle of genome editing. This natural process can be manipulated experimentally or therapeutically to modify the cell’s endogenous genome, making genome editing both a powerful research tool and potential therapeutic modality.

The basis on which a DSB increases genome editing by this enormous magnitude is that a genomic DNA DSB is a dangerous event to the cell. It causes the loss of chromosomal integrity (it separates the telomere from the centromere). Dozens of DSBs occur spontaneously in all living cells per day, caused by variety of different genomic insults. Cells have developed redundant mechanisms to respond to and repair DSBs. The two most prominent are nonhomologous end joining (NHEJ) and homologous recombination (HR).[8,9]

In NHEJ, a complex of proteins holds the broken ends together while the ends are processed to allow ligases to seal the two ends back together. Conceptually, the ends are stitched back together. In general, the NHEJ process is quite accurate, with the two ends being joined precisely 70% of the time, but in the remaining instances the ends are joined after the loss or gain of nucleotides at the site of the break (insertions and deletions [indels]). The NHEJ repair pathway is active throughout the cell cycle.[10] Inactivation of genes needed for NHEJ leads to genomic instability but not to immediate cell death.[11,12]

HR is a biochemically more complex process in which a much larger number of proteins are involved. In mitotic mammalian cells, the mechanism by which cells repair DSBs by HR is through the “synthesis-dependent strand annealing” (SDSA) mechanism (Fig. 1).[13,14] Conceptually, the cell uses an undamaged homologous piece of DNA as a template to synthesize new DNA and then uses the newly synthesized DNA to paste into the damaged DNA to fix the DSB. It can be thought of as a “copy and paste” mechanism. An important feature of the SDSA mechanism of repair is that the template DNA is not physically transferred into the break. Instead, it is the information in the template DNA that the recombination machinery copies and transfers into the site of the break. The normal and natural template DNA is the sister chromatid. The HR repair pathway is thought to be active only in S/G2 of the cell cycle (when a sister chromatid is present). Mutations in important HR genes can cause cell lethality within a single round of replication.[15]

Fig1. OVERVIEW OF NUCLEASE-BASED GENOME EDITING

References

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[1] Smithies O, et al. Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature. 1985;317(6034):230–234.

[2] Sedivy JM, Sharp PA. Positive genetic selection for gene disruption in mammalian cells by homologous recombination. Proc Natl Acad Sci USA. 1989;86:227–231.

[3] Doetschman T, Maeda N, Smithies O. Targeted mutation of the Hprt gene in mouse embryonic stem cells. Proc Natl Acad Sci USA. 1988;85(22):8583 8587.

[4] Thomas KR, Capecchi MR. Introduction of homologous DNA sequences into mammalian cells induces mutations in the cognate gene. Nature. 1986;324(6092):34–38.

 [5] Rouet P, Smih F,M. Jasin, Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol Cell Biol. 1994;14(12):8096–8106.

[6] Choulika A, et al. Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol Cell Biol. 1995;15(4):1968–1973.

 [7] Sargent RG, Brenneman MA, Wilson JH. Repair of site-specific double strand breaks in a mammalian chromosome by homologous and illegitimate recombination. Mol Cell Biol. 1997;17(1):267–277.

[8] Wyman C, Kanaar R. DNA double-strand break repair: all’s well that ends well. Annu Rev Genet. 2006;40:363–383.

[9] West SC, et al. Double-strand break repair in human cells. Cold Spring Harb Symp Quant Biol. 2000;65:315–321.

 [10] Davis AJ, Chen BP, Chen DJ. DNA-PK: a dynamic enzyme in a versatile DSB repair pathway. DNA Repair (Amst). 2014;17:21–29.

 [11] Burma S, Chen BP, Chen DJ. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair (Amst). 2006;5(9-10):1042 1048.

[12] Mills KD, Ferguson DO, Alt FW. The role of DNA breaks in genomic instability and tumorigenesis. Immunol Rev. 2003;194:77–95.

 [13] Xue C, Greene EC. DNA repair pathway choices in CRISPR-Cas9 mediated genome editing. Trends Genet. 2021;37(7):639–656.

[14] Paques F, Haber JE. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 1999;63(2):349–404.

[15] Lim DS, Hasty P. A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53. Mol Cell Biol. 1996;16(12):7133–7143.

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Using Engineered Nucleases to Initiate the Genome Editing Process

Base Editing

Plaque and Colony Hybridization for Clone Identification

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Plasmid Vectors

كلونة النعجة دولي Cloning Dolly the sheep

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أدوات كلونة الجين المتعاقبة

genetic engineering in industry

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البصمة المعتمدة على التغاير العددي في تنوعات التكرارات المترادفةVariable number of tandem repeat polymorphisms