GUIDE-Seq

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GUIDE-Seq (Genome-wide, Unbiased Identification of DSBs Enabled by Sequencing) is a molecular biology technique that allows for the unbiased in vitro detection of off-target genome editing events in DNA caused by CRISPR/Cas9 as well as other RNA-guided nucleases in living cells. [1] Similar to LAM-PCR, it employs multiple PCRs to amplify regions of interest that contain a specific insert that preferentially integrates into double-stranded breaks. As gene therapy is an emerging field, GUIDE-Seq has gained traction as a cheap method to detect the off-target effects of potential therapeutics without needing whole genome sequencing.[ citation needed ]

Contents

Principles

Conceived to work in concert with next-gen sequencing platforms such as Illumina dye sequencing, GUIDE-Seq relies on the integration of a blunt, double-stranded oligodeoxynucleotide (dsODN) that has been phosphothioated on two of the phosphate linkages on the 5' end of both strands. [1] The dsODN cassette integrates into any site in the genome that contains a double-stranded break (DSB). [1] This means that along with the target and off-target sites that may exist as a result of the activity of a nuclease, the dsODN cassette will also integrate into any spurious sites in the genome that have a DSB. [1] This makes it critical to have a dsODN only condition that controls for errant and naturally occurring DSBs, and is required to use the GUIDE-seq bioinformatic pipeline. [1]

After integration of the dsODN cassette, genomic DNA (gDNA) is extracted from the cell culture and sheared to 500bp fragments via sonication. The resulting sheared gDNA undergoes end-repair and adapter ligation. From here, DNA specifically containing the dsODN insert is amplified via two rounds of polymerase chain reaction (PCR) that proceeds in a unidirectional manner starting from the primers that are complementary to the dsODN. This process allows for the reading of the adjacent sequences, both the sense and anti-sense strands, flanking the insert. The final product is a panoply of amplicons, describing the DSB distribution, containing indices for sample differentiation, p5 and p7 Illumina flow-cell adapters, and the sequences flanking the dsODN cassette. [1]

GUIDE-Seq is able to achieve detection of rare DSBs that occur with a 0.1% frequency, however this may be as a result of the limitations of next-generation sequencing platforms. The greater the depth of reads an instrument is able to achieve, the better it can detect rarer events. [1] Additionally, GUIDE-Seq is able to detect sites not predicted by the "in silico" methods which often will predict sites based on sequence similarity and percent mismatch. [1] There have been cases of GUIDE-Seq not detecting any off-targets for certain guide RNAs, suggesting that some RNA-guided nucleases may have no associated off-targets. [1] [2] GUIDE-Seq has been used to show that engineered variants of Cas9 can have reduced off-target effects. [3]

Caveats

GUIDE-Seq has been shown to miss some off-targets, when compared to the genome-wide sequencing DIGENOME-Seq method, due to the nature of its targeting. [4] Another caveat is that GUIDE-Seq has been observed to generate slightly different off-target sites depending on the cell line. [1] This could be due to cell lines having different parental genetic origins, cell line specific mutations, or, in the case of some immortal cell lines such as K562s, having aneuploidy. This suggests that it would be pertinent for researchers to test multiple cell lines to validate efficacy and accuracy. [5] GUIDE-Seq cannot be used to identify off-targets in vivo.

Related Research Articles

A gene knockout is a genetic technique in which one of an organism's genes is made inoperative. However, KO can also refer to the gene that is knocked out or the organism that carries the gene knockout. Knockout organisms or simply knockouts are used to study gene function, usually by investigating the effect of gene loss. Researchers draw inferences from the difference between the knockout organism and normal individuals.

<span class="mw-page-title-main">CRISPR</span> Family of DNA sequence found in prokaryotic organisms

CRISPR is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. These sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote. They are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence these sequences play a key role in the antiviral defense system of prokaryotes and provide a form of acquired immunity. CRISPR is found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea.

Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms. Alongside CRISPR/Cas9 and TALEN, ZFN is a prominent tool in the field of genome editing.

<i>Fok</i>I

The restriction endonuclease Fok1, naturally found in Flavobacterium okeanokoites, is a bacterial type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and a non-specific DNA cleavage domain at the C-terminal. Once the protein is bound to duplex DNA via its DNA-binding domain at the 5'-GGATG-3' recognition site, the DNA cleavage domain is activated and cleaves, without further sequence specificity, the first strand 9 nucleotides downstream and the second strand 13 nucleotides upstream of the nearest nucleotide of the recognition site.

ChIP-sequencing, also known as ChIP-seq, is a method used to analyze protein interactions with DNA. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global binding sites precisely for any protein of interest. Previously, ChIP-on-chip was the most common technique utilized to study these protein–DNA relations.

<span class="mw-page-title-main">Transcription activator-like effector nuclease</span>

Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain. Transcription activator-like effectors (TALEs) can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. The restriction enzymes can be introduced into cells, for use in gene editing or for genome editing in situ, a technique known as genome editing with engineered nucleases. Alongside zinc finger nucleases and CRISPR/Cas9, TALEN is a prominent tool in the field of genome editing.

<span class="mw-page-title-main">Genome editing</span> Type of genetic engineering

Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly inserts genetic material into a host genome, genome editing targets the insertions to site-specific locations. The basic mechanism involved in genetic manipulations through programmable nucleases is the recognition of target genomic loci and binding of effector DNA-binding domain (DBD), double-strand breaks (DSBs) in target DNA by the restriction endonucleases, and the repair of DSBs through homology-directed recombination (HDR) or non-homologous end joining (NHEJ).

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

<span class="mw-page-title-main">Cas9</span> Microbial protein found in Streptococcus pyogenes M1 GAS

Cas9 is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic engineering applications. Its main function is to cut DNA and thereby alter a cell's genome. The CRISPR-Cas9 genome editing technique was a significant contributor to the Nobel Prize in Chemistry in 2020 being awarded to Emmanuelle Charpentier and Jennifer Doudna.

<span class="mw-page-title-main">CRISPR interference</span> Genetic perturbation technique

CRISPR interference (CRISPRi) is a genetic perturbation technique that allows for sequence-specific repression of gene expression in prokaryotic and eukaryotic cells. It was first developed by Stanley Qi and colleagues in the laboratories of Wendell Lim, Adam Arkin, Jonathan Weissman, and Jennifer Doudna. Sequence-specific activation of gene expression refers to CRISPR activation (CRISPRa).

A protospacer adjacent motif (PAM) is a 2–6-base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. The PAM is a component of the invading virus or plasmid, but is not found in the bacterial host genome and hence is not a component of the bacterial CRISPR locus. Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence. PAM is an essential targeting component which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by the CRISPR-associated nuclease.

Cas12a is an RNA-guided endonuclease of that forms part of the CRISPR system in some bacteria and is used by scientists to modify DNA. It originates as part of a bacterial immune mechanism, where it serves to destroy the genetic material of viruses and thus protect the cell and colony from viral infection. Cas12a and other CRISPR associated endonucleases are unique in that their use of a guide RNA makes this action highly selective; they only cut DNA when it is adjacent to a certain sequence of nucleotides. In the organisms from which it originates, this guide RNA is a copy of a piece of the genome from a virus that previously infected the cell.

<span class="mw-page-title-main">Surveyor nuclease assay</span>

Surveyor nuclease assay is an enzyme mismatch cleavage assay used to detect single base mismatches or small insertions or deletions (indels).

SCAR-less genome editing Scarless Cas9 Assisted Recombineering (no-SCAR) is an editing method that is able to manipulate the Escherichia coli genome. The system relies on recombineering whereby DNA sequences are combined and manipulated through homologous recombination. No-SCAR is able to manipulate the E. coli genome without the use of the chromosomal markers detailed in previous recombineering methods. Instead, in this method, the λ-Red recombination system facilitates donor DNA integration while Cas9 cleaves double-stranded DNA to counter-select against wild-type cells. Although λ-Red and Cas9 genome editing are widely used technologies, the no-SCAR method is novel in combining the two functions; this technique is able to establish point mutations, gene deletions, and short sequence insertions in several genomic loci with increased efficiency and time sensitivity.

J. Keith Joung is an American pathologist and molecular biologist who holds the Robert B. Colvin Endowed Chair in Pathology at Massachusetts General Hospital and is Professor of Pathology at Harvard Medical School. He is a leading figure in the field of genome editing and has pioneered the development of designer nucleases and sensitive off-target detection methods.

Perturb-seq refers to a high-throughput method of performing single cell RNA sequencing (scRNA-seq) on pooled genetic perturbation screens. Perturb-seq combines multiplexed CRISPR mediated gene inactivations with single cell RNA sequencing to assess comprehensive gene expression phenotypes for each perturbation. Inferring a gene’s function by applying genetic perturbations to knock down or knock out a gene and studying the resulting phenotype is known as reverse genetics. Perturb-seq is a reverse genetics approach that allows for the investigation of phenotypes at the level of the transcriptome, to elucidate gene functions in many cells, in a massively parallel fashion.

Off-target genome editing refers to nonspecific and unintended genetic modifications that can arise through the use of engineered nuclease technologies such as: clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9, transcription activator-like effector nucleases (TALEN), meganucleases, and zinc finger nucleases (ZFN). These tools use different mechanisms to bind a predetermined sequence of DNA (“target”), which they cleave, creating a double-stranded chromosomal break (DSB) that summons the cell's DNA repair mechanisms and leads to site-specific modifications. If these complexes do not bind at the target, often a result of homologous sequences and/or mismatch tolerance, they will cleave off-target DSB and cause non-specific genetic modifications. Specifically, off-target effects consist of unintended point mutations, deletions, insertions inversions, and translocations.

<span class="mw-page-title-main">CRISPR gene editing</span> Gene editing method

CRISPR gene editing is a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified. It is based on a simplified version of the bacterial CRISPR-Cas9 antiviral defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added in vivo.

BLESS, also known as breaks labeling, enrichment on streptavidin and next-generation sequencing, is a method used to detect genome-wide double-strand DNA damage. In contrast to chromatin immunoprecipitation (ChIP)-based methods of identifying DNA double-strand breaks (DSBs) by labeling DNA repair proteins, BLESS utilizes biotinylated DNA linkers to directly label genomic DNA in situ which allows for high-specificity enrichment of samples on streptavidin beads and the subsequent sequencing-based DSB mapping to nucleotide resolution.

<span class="mw-page-title-main">Kim Jin-soo (biologist)</span> Korean scientist

Kim Jin-Soo is a chemist, biologist, and entrepreneur. He was CEO and CSO, ToolGen, Inc., is a professor in the Department of Chemistry of Seoul National University and director of the Center for Genome Engineering. His research team has developed and improved several types of programmable nucleases, specifically zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs). In 2018, he was a Clarivate Analytics Highly Cited Researcher in the cross-field category and in the biology and biochemistry category in 2019.

References

  1. 1 2 3 4 5 6 7 8 9 10 Tsai, Shengdar Q.; Zheng, Zongli; Nguyen, Nhu T.; Liebers, Matthew; Topkar, Ved V.; Thapar, Vishal; Wyvekens, Nicolas; Khayter, Cyd; Iafrate, A. John; Le, Long P.; Aryee, Martin J.; Joung, J. Keith (February 2015). "GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases". Nature Biotechnology. 33 (2): 187–197. doi:10.1038/nbt.3117. PMC   4320685 . PMID   25513782.
  2. ↑ Akcakaya, Pinar; Bobbin, Maggie L.; Guo, Jimmy A.; Malagon-Lopez, Jose; Clement, Kendell; Garcia, Sara P.; Fellows, Mick D.; Porritt, Michelle J.; Firth, Mike A.; Carreras, Alba; Baccega, Tania; Seeliger, Frank; Bjursell, Mikael; Tsai, Shengdar Q.; Nguyen, Nhu T.; Nitsch, Roberto; Mayr, Lorenz M.; Pinello, Luca; Bohlooly-Y, Mohammad; Aryee, Martin J.; Maresca, Marcello; Joung, J. Keith (12 September 2018). "In vivo CRISPR editing with no detectable genome-wide off-target mutations". Nature. 561 (7723): 416–419. Bibcode:2018Natur.561..416A. doi:10.1038/s41586-018-0500-9. PMC   6194229 . PMID   30209390.
  3. ↑ Kleinstiver, Benjamin P.; Pattanayak, Vikram; Prew, Michelle S.; Tsai, Shengdar Q.; Nguyen, Nhu T.; Zheng, Zongli; Joung, J. Keith (January 2016). "High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects". Nature. 529 (7587): 490–495. Bibcode:2016Natur.529..490K. doi:10.1038/nature16526. PMC   4851738 . PMID   26735016.
  4. ↑ Kim, Daesik; Kim, Sojung; Kim, Sunghyun; Park, Jeongbin; Kim, Jin-Soo (March 2016). "Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq". Genome Research. 26 (3): 406–415. doi:10.1101/gr.199588.115. PMC   4772022 . PMID   26786045.
  5. ↑ Alberts, Bruce (2014). Molecular Biology of the Cell (6th ed.). Garland Science.
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