Growing confidence in Template-free CRISPR-induced gene editing

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Genes possess the natural mechanism to repair a broken DNA sequence. However, this repair was conventionally believed to be unpredictable, erroneous and random. Traditionally, gene editing techniques were aimed at replacing a segment of the genetic sequence with a template sequence to achieve the desired genetic modification… (Featured image is for representative purpose only and has been sourced from https://pixabay.com/en/sculpture-chakra-figure-art-metal-2655931/)

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Genes possess the natural mechanism to repair a broken DNA sequence. However, this repair was conventionally believed to be unpredictable, erroneous and random. Traditionally, gene editing techniques were aimed at replacing a segment of the genetic sequence with a template sequence to achieve the desired genetic modification. Known as homology-directed repair (HDR), the method included replacing an undesirable genetic mutation with the correct sequence to get rid of the abnormalities. Like its predecessors, CRISPR gene editing technique was also being developed to bring about gene modification by using a donor template post the snip caused by the Cas9 protein.

According to a new study published in Nature in November 2018 led by Brigham and Women’s Hospital and the Broad Institute of MIT and Harvard, CRISPR-Cas9 is capable of precise genome editing even without the donor DNA templates. Template-free Cas9 editing, as the study demonstrates, is predictable and capable of precise repair to a predicted genotype, enabling correction of disease-associated mutations in humans. Further, the study concludes optimistically that the cell’s genetic auto-correction could be combined with CRISPR-based therapies to correct gene mutations by simply cutting DNA precisely and allowing the cell to naturally heal the damage. While this is a futuristic idea, it seems quite a hopeful possibility.

Earlier in November 2017, researchers at University Hospital Frankfurt, Germany, noted that donor template-free CRISPR editing can effectively repair frameshift mutation in Cytochrome b-245 heavy chain gene (CYBB). Notably, inventors Zengyu Wang and Miao Chen of Noble Research Institute LLC filed a patent application US20180201944A1 claiming priority from January 17th, 2017, claiming a DNA-free genome editing method.

“Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR associated protein 9 (Cas9) offer an effective way of creating targeted mutagenesis in plants. To alleviate concerns related to genetically modified plants, the present disclosure provides a novel and efficient genome editing system that allows the regeneration of mutant plants without DNA stable integration. This DNA free system utilizes Cas9 mRNA, guide RNA and selectable marker RNA to infect plant protoplasts. After a short period of selection of the transfected cells, non-transgenic plants carrying expected mutations were regenerated. The system offers a way of creating desired mutants without transgenic elements.”

However, their pending patent application US20180201944A1 does not specifically claim template free gene editing.

A method for selecting cells carrying a transfected nucleic acid comprising the steps of:

exposing a plurality of cells to a first RNA molecule under conditions sufficient to promote transfection of the first RNA molecule into one or more of the plurality of cells, wherein the first RNA molecule or a peptide or protein encoded by the first RNA molecule permits selection of the one or more of the plurality of cells transfected with the first RNA molecule based on a first selectable property; and

selecting the one or more cells of the plurality of cells transfected with the first RNA molecule based on the first selectable property.

16. The method of claim 1 further comprising exposing the plurality of cells to a second RNA molecule and a third RNA molecule in addition to the first RNA molecule under conditions sufficient to promote transfection of the second RNA molecule and third RNA molecule, wherein the second RNA molecule encodes Cas9, wherein the third RNA molecule is guide RNA (gRNA), wherein the gRNA comprises a scaffold sequence for Cas9 binding and a target sequence, wherein the target sequence defines the genomic region of the plurality of cells to be edited, wherein the second RNA molecule and third RNA molecule are sufficient to edit a genomic region of one or more of the plurality of cells, and wherein the plurality of cells are plant cells. 

Patent Application US20160201089A1 filed by Duke University claiming priority from June 5, 2013, discloses template-free CRISPR based gene editing.

[0274] c. Methods of Correcting a Mutant Gene and Treating a Subject Using CRISPR/Cas9

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 [0276] The present disclosure is directed to genome editing with CRISPR/Cas9-based system without a repair template, which can efficiently correct the reading frame and restore the expression of a functional protein involved in a genetic disease. The disclosed CRISPR/Cas9-based system and methods may involve using homology-directed repair or nuclease-mediated non-homologous end joining (NHEJ)-based correction approaches, which enable efficient correction in proliferation-limited primary cell lines that may not be amenable to homologous recombination or selection-based gene correction. This strategy integrates the rapid and robust assembly of active CRISPR/Cas9-based system with an efficient gene editing method for the treatment of genetic diseases caused by mutations in nonessential coding regions that cause frameshifts, premature stop codons, aberrant splice donor sites or aberrant splice acceptor sites.

Several patent applications have discussed template-free gene editing methods but have considered it to be error prone. While DNA self-repair was already known since decades, template-free CRISPR-induced gene editing technique has drawn research interest in the past year due to predictability and accuracy. Advances in machine learning enable trained mathematical models to accurately predict the sequence generated by self-repair post-CRISPR Cas9 based sequence cleavage. Template-free gene editing is no longer an unpredictable, random and erroneous method of gene editing as believed earlier.

The advantages of relying on the auto-repair technique of the genetic sequences are multifold. Known as Nuclease Mediated Non-Homologous End Joining (NHEJ), this template-free repair method does not require a donor template, eliminating the chances of nonspecific insertional mutagenesis. In contrast to HDR, NHEJ operates effectively and efficiently in all stages of the cell cycle and therefore may be effectively applied in both cycling and post-mitotic cells, such as muscle fibers. This provides a robust, permanent gene restoration alternative opening doors to a plethora of industrial applications in medicine and agriculture.

Ethical and regulatory objections to genetically mutant agricultural produce can be alleviated by allowing genetic sequence to self-modify without the introduction and integration of foreign DNA. Template-free gene editing will also enable fast and efficient ways of correcting genetic disorders. Relying on genes’ inherent ability to repair themselves correctly eliminates the need to introduce donor templates, thereby speeding up the process and making it cost effective.

(Featured image is for representative purpose only and has been sourced from https://pixabay.com/en/sculpture-chakra-figure-art-metal-2655931/)

Aditi Das
Aditi Das

Aditi Das is a compulsive sci-fi dreamer who conjures up aliens from vehicles stuck in a traffic snarl. When she isn’t driving teams, inspiring them to shred technologies to pieces and unearth their DNA, she’s busy dishing up Bengali food and blogging.


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