Non-Homologous End Joining and Homology Directed DNA Repair Frequency of Double-Stranded Breaks Introduced by Genome Editing Reagents

Zaboikin, Michail; Zaboikina, Tatiana; Freter, Carl E.; Srinivasakumar, Narasimhachar

doi:10.1371/journal.pone.0169931

Cited by 58 publications

(51 citation statements)

References 34 publications

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“…This is followed by amplification of target region by PCR and generation of heteroduplexes by denaturation and renaturation in the presence of unmodified wild type or different alleles. Mismatches in these heteroduplexes can be identified by digestion with single-strand specific endonucleases (such as T7 or Surveyor nuclease) and resolution of the digestion products in polyacrylamide or agarose gels [ 10 – 12 ].…”

Section: Introductionmentioning

confidence: 99%

Gaussian decomposition of high-resolution melt curve derivatives for measuring genome-editing efficiency

2018

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We describe a method for measuring genome editing efficiency from in silico analysis of high-resolution melt curve data. The melt curve data derived from amplicons of genome-edited or unmodified target sites were processed to remove the background fluorescent signal emanating from free fluorophore and then corrected for temperature-dependent quenching of fluorescence of double-stranded DNA-bound fluorophore. Corrected data were normalized and numerically differentiated to obtain the first derivatives of the melt curves. These were then mathematically modeled as a sum or superposition of minimal number of Gaussian components. Using Gaussian parameters determined by modeling of melt curve derivatives of unedited samples, we were able to model melt curve derivatives from genetically altered target sites where the mutant population could be accommodated using an additional Gaussian component. From this, the proportion contributed by the mutant component in the target region amplicon could be accurately determined. Mutant component computations compared well with the mutant frequency determination from next generation sequencing data. The results were also consistent with our earlier studies that used difference curve areas from high-resolution melt curves for determining the efficiency of genome-editing reagents. The advantage of the described method is that it does not require calibration curves to estimate proportion of mutants in amplicons of genome-edited target sites.

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Section: Introductionmentioning

confidence: 99%

Gaussian decomposition of high-resolution melt curve derivatives for measuring genome-editing efficiency

2018

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“…Even though NHEJ is vilified in the world of precise genome engineering as being error-prone, it is actually an overstatement. Cas9 cleavage of target site is invariably followed by NHEJ repair, and the process is repeated until there is an error, which prevents Cas9-mediated DNA cleavage ( 53 ). Thus, the end-product of NHEJ observed might be an error, although NHEJ intrinsically does not result in such errors.…”

Section: Manipulating Cell Cycle To Enhance Hdr In Non-dividing Cellsmentioning

confidence: 99%

The road less traveled: strategies to enhance the frequency of homology-directed repair (HDR) for increased efficiency of CRISPR/Cas-mediated transgenesis

Devkota

2018

BMB Rep.

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Non-homologous end joining (NHEJ), and to a lesser extent, the error-free pathway known as homology-directed repair (HDR) are cellular mechanisms for recovery from double-strand DNA breaks (DSB) induced by RNA-guided programmable nuclease CRISPR/Cas. Since NHEJ is equivalent to using a duck tape to stick two pieces of metals together, the outcome of this repair mechanism is prone to error. Any out-of-frame mutations or premature stop codons resulting from NHEJ repair mechanism are extremely handy for loss-of-function studies. Substitution of a mutation on the genome with the correct exogenous repair DNA requires coordination via an error-free HDR, for targeted transgenesis. However, several practical limitations exist in harnessing the potential of HDR to replace a faulty mutation for therapeutic purposes in all cell types and more so in somatic cells. In germ cells after the DSB, copying occurs from the homologous chromosome, which increases the chances of incorporation of exogenous DNA with some degree of homology into the genome compared with somatic cells where copying from the identical sister chromatid is always preferred. This review summarizes several strategies that have been implemented to increase the frequency of HDR with a focus on somatic cells. It also highlights the limitations of this technology in gene therapy and suggests specific solutions to circumvent those barriers.

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“…For some experiments we used a different forward primer, SK214, that was located further upstream and produced a PCR product of size 140 bp with reverse primer SK145. The sequences and genome locations of these primers have been described earlier (12). The gDNA from unmodified or mocktransfected cells were also amplified in parallel using the same primer pairs.…”

Section: Amplification Of Target Loci For Obtaining High-resolution Mmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Gaussian decomposition of high-resolution melt curve derivatives for measuring genome-editing efficiency

Zaboikin

Freter

Srinivasakumar

2017

Preprint

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24We describe a method for measuring genome editing efficiency from in silico analysis of 25 high-resolution melt curve data. The melt curve data derived from amplicons of genome-26 edited or unmodified target sites were processed to remove the background fluorescent 27 signal emanating from free fluorophore and then corrected for temperature-dependent 28 quenching of fluorescence of double-stranded DNA-bound fluorophore. Corrected data 29 were normalized and numerically differentiated to obtain the first derivatives of the melt 30 curves. These were then mathematically modeled as a sum or superposition of minimal 31 number of Gaussian components. Using Gaussian parameters determined by modeling of 32 melt curve derivatives of unedited samples, we were able to model melt curve derivatives 33 from genetically altered target sites where the mutant population could be accommodated 34 using an additional Gaussian component. From this, the proportion contributed by the 35 mutant component in the target region amplicon could be accurately determined. Mutant 36 component computations compared well with the mutant frequency determination from 37 next generation sequencing data. The results were also consistent with our earlier studies 38 that used difference curve areas from high-resolution melt curves for determining the 39 efficiency of genome-editing reagents. The advantage of the described method is that it 40 does not require calibration curves to estimate proportion of mutants in amplicons of 41 genome-edited target sites. 42Homology-directed repair (HDR) either restores the original in the presence of an 49 endogenous template (sister chromatid) or inserts an exogenous DNA donor template 50 when available across the cut site [7][8][9]. 51The ability to generate genome-editing reagents with a desired specificity does not 52 guarantee efficient target site modification. There is therefore a need for methods that 53 rapidly assess reagent efficacy. A common approach is to determine efficacy of genome 54 editing reagents is to transfect human embryonic kidney (HEK293T) cell line with the 55 reagents. This is followed by amplification of target region by PCR and generation of 56 heteroduplexes by denaturation and renaturation in the presence of unmodified wild type 57 or different alleles. Mismatches in these heteroduplexes can be identified by digestion with 58 single-strand specific endonucleases (such as T7 or Surveyor nuclease) and resolution of 59 the digestion products in polyacrylamide or agarose gels [10][11][12]. 60A second approach to determine efficacy of genome editing is to use TaqMan assays with 61 probes designed to bind over the putative target cut site [12,13]. Reduced binding of the 62 TaqMan probe, due to indel mutations at the target site, with reference to a control 63TaqMan probe that binds outside the cut site, can be used to estimate the editing efficacy. 64 A third method, which is gaining popularity, uses high resolution melting analysis (HRMA) 65 after real-time PCR with nonspecific double-st...

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Non-Homologous End Joining and Homology Directed DNA Repair Frequency of Double-Stranded Breaks Introduced by Genome Editing Reagents

Cited by 58 publications

References 34 publications

Gaussian decomposition of high-resolution melt curve derivatives for measuring genome-editing efficiency

Gaussian decomposition of high-resolution melt curve derivatives for measuring genome-editing efficiency

The road less traveled: strategies to enhance the frequency of homology-directed repair (HDR) for increased efficiency of CRISPR/Cas-mediated transgenesis

Gaussian decomposition of high-resolution melt curve derivatives for measuring genome-editing efficiency

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