Genome-wide CRISPR-Cas9 knockout screens

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Genome-wide CRISPR-Cas9 knockout screens aim to elucidate the relationship between genotype and phenotype by ablating gene expression on a genome-wide scale and studying the resulting phenotypic alterations. The approach utilises the CRISPR-Cas9 gene editing system, coupled with libraries of single guide RNAs (sgRNAs), which are designed to target every gene in the genome. Over recent years, the genome-wide CRISPR screen has emerged as a powerful tool for performing large-scale loss-of-function screens, with low noise, high knockout efficiency and minimal off-target effects.

Contents

Genome-wide CRISPR/Cas9 Knockout Screens: Workflow Overview. 1. sgRNA Library Creation: sgRNAs are computationally designed, synthesised, amplified by PCR, and cloned into a vector delivery system. This can be skipped by obtaining a commercially available library. 2. Screen: Cells are transduced with lentiviral particles containing the sgRNA library, Cas9, and other necessary components. Cells may be transduced in bulk (pooled screen), or in individual wells (arrayed screen). 3: Measurement & Analysis. Desired clones are selected and DNA is extracted. In a pooled screen, DNA sequencing will be necessary. sgRNAs are recovered, analysed, and associated genes identified. Genome-wide CRISPR screen - Overview.png
Genome-wide CRISPR/Cas9 Knockout Screens: Workflow Overview. 1. sgRNA Library Creation: sgRNAs are computationally designed, synthesised, amplified by PCR, and cloned into a vector delivery system. This can be skipped by obtaining a commercially available library. 2. Screen: Cells are transduced with lentiviral particles containing the sgRNA library, Cas9, and other necessary components. Cells may be transduced in bulk (pooled screen), or in individual wells (arrayed screen). 3: Measurement & Analysis. Desired clones are selected and DNA is extracted. In a pooled screen, DNA sequencing will be necessary. sgRNAs are recovered, analysed, and associated genes identified.

History

Early studies in Caenorhabditis elegans [1] and Drosophila melanogaster [2] [3] saw large-scale, systematic loss of function (LOF) screens performed through saturation mutagenesis, demonstrating the potential of this approach to characterise genetic pathways and identify genes with unique and essential functions. The saturation mutagenesis technique was later applied in other organisms, for example zebrafish [4] [5] and mice. [6] [7]

Targeted approaches for gene knockdown emerged in the 1980s with techniques such as homologous recombination, [8] [9] trans-cleaving ribozymes, [10] [11] and antisense technologies. [12] [13]

By the year 2000, RNA interference (RNAi) technology had emerged as a fast, simple, and inexpensive technique for targeted gene knockdown, and was routinely being used to study in vivo gene function in C. elegans. [14] [15] [16] [17] Indeed, in the span of only a few years following its discovery by Fire et al. (1998), [18] almost all of the ~19,000 genes in C. elegans had been analysed using RNAi-based knockdown. [19]

The production of RNAi libraries facilitated the application of this technology on a genome-wide scale, and RNAi-based methods became the predominant approach for genome-wide knockdown screens.[ citation needed ]

Nevertheless, RNAi-based approaches to genome-wide knockdown screens have their limitations. For one, the high off-target effects cause issues with false-positive observations. [20] [21] Additionally, because RNAi reduces gene expression at the post-transcriptional level by targeting RNA, RNAi-based screens only result in partial and short-term suppression of genes. Whilst partial knockdown may be desirable in certain situations, a technology with improved targeting efficiency and fewer off-target effects was needed.[ citation needed ]

Since initial identification as a prokaryotic adaptive immune system, [22] the bacterial type II clustered regularly interspaced short palindrome repeats (CRISPR)/Cas9 system has become a simple and efficient tool for generating targeted LOF mutations. [23] It has been successfully applied to edit human genomes, and has started to displace RNAi as the dominant tool in mammalian studies. [24] In the context of genome-wide knockout screens, recent studies have demonstrated that CRISPR/Cas9 screens are able to achieve highly efficient and complete protein depletion, and overcome the off-target issues seen with RNAi screens. [25] [26] In summary, the recent emergence of CRISPR-Cas9 has dramatically increased our ability to perform large-scale LOF screens. The versatility and programmability of Cas9, coupled with the low noise, high knockout efficiency and minimal off-target effects, have made CRISPR the platform of choice for many researchers engaging in gene targeting and editing. [24] [27]

Methods

CRISPR/Cas9 Loss of function

The CRISPR-Cas9 gene editing mechanism. Cas9 cleaves dsDNA upstream (5') of the PAM site (red), and the gRNA provides a template for repair. GRNA-Cas9.png
The CRISPR-Cas9 gene editing mechanism. Cas9 cleaves dsDNA upstream (5') of the PAM site (red), and the gRNA provides a template for repair.

The clustered regularly interspaced short palindrome repeats (CRISPR)/Cas9 system is a gene-editing technology that can introduce double-strand breaks (DSBs) at a target genomic locus. By using a single guide RNA (sgRNA), the endonuclease Cas9 can be delivered to a specific DNA sequence where it cleaves the nucleotide chain. [28] The specificity of the sgRNA is determined by a 20-nt sequence, homologous to the genomic locus of interest, and the binding to Cas9 is mediated by a constant scaffold region of the sgRNA. The desired target site must be immediately followed (5’ to 3’) by a conserved 3 nucleotide protospacer adjacent motif (PAM). [29] [30] In order to repair the DSBs, the cell may use the highly error prone non-homologous end joining, or homologous recombination. By designing suitable sgRNAs, planned insertions or deletions can be introduced into the genome. In the context of genome-wide LOF screens, the aim is to cause gene disruption and knockout.[ citation needed ]

sgRNA libraries

Constructing a Library

To perform CRISPR knockouts on a genome-wide scale, collections of sgRNAs known as sgRNA libraries, or CRISPR knockout libraries, must be generated. The first step in creating a sgRNA library is to identify genomic regions of interest based on known sgRNA targeting rules. [31] For example, sgRNAs are most efficient when targeting the coding regions of genes and not the 5’ and 3’ UTRs. Conserved exons present as attractive targets, and position relative to the transcription start site should be considered. [31] Secondly, all the possible PAM sites are identified and selected for. [31] On- and off-target activity should be analysed, as should GC content, and homopolymer stretches should be avoided. [31] The most commonly used Cas9 endonuclease, derived from Streptococcus pyogenes , recognises a PAM sequence of NGG. [32]

Furthermore, specific nucleotides appear to be favoured at specific locations. Guanine is strongly favoured over cytosine on position 20 right next to the PAM motif, and on position 16 cytosine is preferred over guanine. [33] For the variable nucleotide in the NGG PAM motif, it has been shown that cytosine is preferred and thymine disfavoured. [33] With such criteria taken into account, the sgRNA library is computationally designed around the selected PAM sites. [31] [33] [34]

Multiple sgRNAs (at least 4–6) should be created against every single gene to limit false-positive detection, and negative control sgRNAs with no known targets should be included. [31] [33] The sgRNAs are then created by in situ synthesis, amplified by PCR, and cloned into a vector delivery system.

Existing libraries

Developing a new sgRNA library is a laborious and time-consuming process. In practice, researchers may select an existing library depending on their experimental purpose and cell lines of interest. As of February 2020, the most widely used resources for genome-wide CRISPR knockout screens have been the two Genome-Scale CRISPR Knock-Out (GeCKO) libraries created by the Zhang lab. [35] Available through Addgene, these lentiviral libraries respectively target human and mouse exons, and both are available as a one-vector system (where the sgRNAs and Cas9 are present on the same plasmid) or as a two-vector system (where the sgRNAs and Cas9 are present on separate plasmids). Each library is delivered as two half-libraries, allowing researchers to screen with 3 or 6 sgRNAs/gene. [36]

Aside from GeCKO, a number of other CRISPR libraries have been generated and made available through Addgene. The Sabatini & Lander labs currently have 7 separate human and mouse libraries, including targeted sublibraries for distinct subpools such as kinases and ribosomal genes (Addgene #51043–51048). Further, improvements to the specificity of sgRNAs have resulted in ‘second generation’ libraries, such as the Brie (Addgene #73632) and Brunello (Addgene #73178) libraries generated by the Doench and Root labs, and the Toronto knockout (TKO) library (Addgene #1000000069) generated by the Moffat lab. [36]

Lentiviral vectors

Producing Infectious Transgenic Lentivirus: A Simple Schematic. Two transfer plasmids, encoding Cas9 and sgRNA separately, may be used depending on the applied library. Producing Infectious Transgenic Lentivirus.png
Producing Infectious Transgenic Lentivirus: A Simple Schematic. Two transfer plasmids, encoding Cas9 and sgRNA separately, may be used depending on the applied library.

Targeted gene knockout using CRISPR/Cas9 requires the use of a delivery system to introduce the sgRNA and Cas9 into the cell. Although a number of different delivery systems are potentially available for CRISPR, [37] [38] genome-wide loss-of-function screens are predominantly carried out using third generation lentiviral vectors. [35] [39] [40] These lentiviral vectors are able to efficiently transduce a broad range of cell types and stably integrate into the genome of dividing and non-dividing cells. [41] [42] Third generation lentiviral particles are produced by co-transfecting 293T human embryonic kidney (HEK) cells with:

  1. two packaging plasmids, one encoding Rev and the other Gag and Pol;
  2. an interchangeable envelope plasmid that encodes for an envelope glycoprotein of another virus (most commonly the G protein of vesicular stomatitis virus (VSV-G));
  3. one or two (depending on the applied library) transfer plasmids, encoding for Cas9 and sgRNA, as well as selection markers. [35] [43] [44]

The lentiviral particle-containing supernatant is harvested, concentrated and subsequently used to infect the target cells. [45] The exact protocol for lentiviral production will vary depending on the research aim and applied library. [35] [43] [44] If a two vector-system is used, for example, cells are sequentially transduced with Cas9 and sgRNA in a two-step procedure. [35] [44] Although more complex, this has the advantage of a higher titre for the sgRNA library virus. [35]

Phenotypic selection

In general, there are two different formats of genome-wide CRISPR knockout screens: arrayed and pooled. In an arrayed screen, each well contains a specific and known sgRNA targeting a specific gene. [46] Since the sgRNA responsible for each phenotype is known based on well location, phenotypes can be identified and analysed without requiring genetic sequencing. This format allows for the measurement of more specific cellular phenotypes, perhaps by fluorescence or luminescence, and allows researchers to use more library types and delivery methods. [46] For large-scale LOF screens, however, arrayed formats are considered low-efficiency, and expensive in terms of financial and material resources because cell populations have to be isolated and cultured individually. [46]

In a pooled screen, cells grown in a single vessel are transduced in bulk with viral vectors collectively containing the entire sgRNA library. To ensure that the amount of cells infected by more than one sgRNA-containing particle is limited, a low multiplicity of infection (MOI) (typically 0.3-0.6) is used. [46] [47] Evidence so far has suggested that each sgRNA should be represented in a minimum of 200 cells. [48] [23] Transduced cells will be selected for, followed by positive or negative selection for the phenotype of interest, and genetic sequencing will be necessary to identify the integrated sgRNAs. [46]

Next-generation sequencing & hit analysis

Following phenotypic selection, genomic DNA is extracted from the selected clones, alongside a control cell population. [23] [46] [49] In the most common protocols for genome-wide knockouts, a 'Next-generation sequencing (NGS) library' is created by a two step polymerase chain reaction (PCR). [23] [46] The first step amplifies the sgRNA region, using primers specific to the lentiviral integration sequence, and the second step adds Illumina i5 and i7 sequences. [23] NGS of the PCR products allows the recovered sgRNAs to be identified, and a quantification step can be used to determine the relative abundance of each sgRNA. [23]

The final step in the screen is to computationally evaluate the significantly enriched or depleted sgRNAs, trace them back to their corresponding genes, and in turn determine which genes and pathways could be responsible for the observed phenotype. Several algorithms are currently available for this purpose, with the most popular being the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) method. [50] Developed specifically for CRISPR/Cas9 knockout screens in 2014, MAGeCK demonstrated better performance compared with alternative algorithms at the time, [50] and has since demonstrated robust results and high sensitivity across different experimental conditions. [51] As of 2015, the MAGeCK algorithm has been extended to introduce quality control measurements, and account for the previously overlooked sgRNA knockout efficiency. [51] A web-based visualisation tool (VISPR) was also integrated, allowing users to interactively explore the results, analysis, and quality controls. [51]

Applications

Cellular signaling mechanisms

Over recent years, the genome-wide CRISPR screen has emerged as a powerful tool for studying the intricate networks of cellular signaling. [52] Cellular signaling is essential for a number of fundamental biological processes, including cell growth, proliferation, differentiation, and apoptosis.

One practical example is the identification of genes required for proliferative signaling in cancer cells. Cells are transduced with a CRISPR sgRNA library, and studied for growth over time. By comparing sgRNA abundance in selected cells to a control, one can identify which sgRNAs become depleted and in turn which genes may be responsible for the proliferation defect. Such screens have been used to identify cancer-essential genes in acute myeloid leukemia [53] and neuroblastoma, [54] and to describe tumor-specific differences between cancer cell lines. [55]

Identifying synthetic lethal partners

Targeted cancer therapies are designed to target the specific genes, proteins, or environments contributing to tumor cell growth or survival. After a period of prolonged treatment with these therapies, however, tumor cells may develop resistance. Although the mechanisms behind cancer drug resistance are poorly understood, potential causes include: target alteration, drug degradation, apoptosis escape, and epigenetic alterations. [56] Resistance is well-recognised and poses a serious problem in cancer management.[ citation needed ]

To overcome this problem, a synthetic lethal partner can be identified. Genome-wide LOF screens using CRISPR-Cas9 can be used to screen for synthetic lethal partners. [57] For this, a wild-type cell line and a tumor cell line containing the resistance-causing mutation are transduced with a CRISPR sgRNA library. The two cell lines are cultivated, and any under-represented or dead cells are analyzed to identify potential synthetic lethal partner genes. A recent study by Hinze et al. (2019) [58] used this method to identify a synthetic lethal interaction between the chemotherapy drug asparaginase and two genes in the Wnt signalling pathway NKD2 and LGR6.

Host dependency factors for viral infection

Due to their small genomes and limited number of encoded proteins, viruses exploit host proteins for entry, replication, and transmission. Identification of such host proteins, also termed host dependency factors (HDFs), is particularly important for identifying therapeutic targets. Over recent years, many groups have successfully used genome-wide CRISPR/Cas9 as a screening strategy for HDFs in viral infections. [59]

One example is provided by Marceau et al. (2017), [60] who aimed to dissect the host factors associated with dengue and hepatitis C (HCV) infection (two viruses in family Flaviviridae). ELAVL1, an RNA-binding protein encoded by the ELAVL1 gene, was found to be a critical receptor for HCV entry, and a remarkable divergence in host dependency factors was demonstrated between the two flaviviridae. [60]

Further applications

Additional reported applications of genome-wide CRISPR screens include the study of: mitochondrial metabolism, [61] bacterial toxin resistance, [62] genetic drivers of metastasis, [63] cancer drug resistance, [64] West Nile virus-induced cell death, [65] and immune cell gene networks. [66] [67]

Limitations

This section will specifically address genome-wide CRISPR screens. For a review of CRISPR limitations see Lino et al. (2018) [38]

The sgRNA library

Genome-wide CRISPR screens will ultimately be limited by the properties of the chosen sgRNA library. Each library will contain a different set of sgRNAs, and average coverage per gene may vary. Currently available libraries tend to be biased towards sgRNAs targeting early (5’) protein-coding exons, rather than those targeting the more functional protein domains. [58] This problem was highlighted by Hinze et al. (2019), [58] who noted that genes associated with asparaginase sensitivity failed to score in their genome-wide screen of asparaginase-resistant leukemia cells.

If an appropriate library is not available, creating and amplifying a new sgRNA library is a lengthy process which may take many months. Potential challenges include: (i) effective sgRNA design; (ii) ensuring comprehensive sgRNA coverage throughout the genome; (iii) lentiviral vector backbone design; (iv) producing sufficient amounts of high-quality lentivirus; (v) overcoming low transformation efficiency; (vi) proper scaling of the bacterial culture. [68]

Maintaining cellular sgRNA coverage

One of the largest hurdles for genome-wide CRISPR screening is ensuring adequate coverage of the sgRNA library across the cell population. [23] Evidence so far has suggested that each sgRNA should be represented and maintained in a minimum of 200-300 cells. [23] [48]

Considering that the standard protocol uses a multiplicity of infection of ~0.3, and a transduction efficiency of 30-40% [44] [23] the number of cells required to produce and maintain suitable coverage becomes very large. By way of example, the most popular human sgRNA library is the GeCKO v2 library created by the Zhang lab; [30] it contains 123,411 sgRNAs. Studies using this library commonly transduce more than 1x108 cells [58] [59] [69]

As CRISPR continues to exhibit low noise and minimal off-target effects, an alternative strategy is to reduce the number of sgRNAs per gene for a primary screen. Less stringent cut-offs are used for hit selection, and additional sgRNAs are later used in a more specific secondary screen. This approach is demonstrated by Doench et al. (2016), [33] who found that >92% of genes recovered using the standard protocol were also recovered using fewer sgRNAs per gene. They suggest that this strategy could be useful in studies where scale-up is prohibitively costly.[ citation needed ]

Lentiviral limitations

Lentiviral vectors have certain general limitations. For one, it is impossible to control where the viral genome integrates into the host genome, and this may affect important functions of the cell. Vannucci et al. [70] provide an excellent review of viral vectors along with their general advantages and disadvantages. In the specific context of genome-wide CRISPR screens, producing and transducing the lentiviral particles is relatively laborious and time-consuming, taking about two weeks in total. [44] Additionally, because the DNA integrates into the host genome, lentiviral delivery leads to long-term expression of Cas9, potentially leading to off-target effects.[ citation needed ]

Arrayed vs pooled screens

In an arrayed screen, each well contains a specific and known sgRNA targeting a specific gene. Arrayed screens therefore allow for detailed profiling of a single cell, but are limited by high costs and the labour required to isolate and culture the high number of individual cell populations. [46] Conventional pooled CRISPR screens are relatively simple and cost effective to perform, but are limited to the study of the entire cell population. This means that rare phenotypes may be more difficult to identify, and only crude phenotypes can be selected for e.g. cell survival, proliferation, or reporter gene expression.[ citation needed ]

Culture media

The choice of culture medium might affect the physiological relevance of findings from cell culture experiments due to the differences in the nutrient composition and concentrations. [71] A systematic bias in generated datasets was recently shown for CRISPR and RNAi gene silencing screens (especially for metabolic genes), [72] and for metabolic profiling of cancer cell lines. [71]  For example, a stronger dependence on ASNS (asparagine synthetase) was found in cell lines cultured in DMEM, which lacks asparagine, compared to cell lines cultured in RPMI or F12 (containing asparagine). [72] Avoiding such bias might be achieved by using a uniform media for all screened cell lines, and ideally, using a growth medium that better represents the physiological levels of nutrients. Recently, such media types, as Plasmax [73] and Human Plasma Like Medium (HPLM), [74] were developed. 

Future Directions

CRISPR + single cell RNA-seq

Emerging technologies are aiming to combine pooled CRISPR screens with the detailed resolution of massively parallel single-cell RNA-sequencing (RNA-seq). Studies utilising “CRISP-seq”, [75] “CROP-seq”, [76] and “PERTURB-seq” [77] have demonstrated rich genomic readouts, accurately identifying gene expression signatures for individual gene knockouts in a complex pool of cells. These methods have the added benefit of producing transcriptional profiles of the sgRNA-induced cells. [78]

Related Research Articles

Gene knockouts are a widely used genetic engineering technique that involves the targeted removal or inactivation of a specific gene within an organism's genome. This can be done through a variety of methods, including homologous recombination, CRISPR-Cas9, and TALENs.

A genetic screen or mutagenesis screen is an experimental technique used to identify and select individuals who possess a phenotype of interest in a mutagenized population. Hence a genetic screen is a type of phenotypic screen. Genetic screens can provide important information on gene function as well as the molecular events that underlie a biological process or pathway. While genome projects have identified an extensive inventory of genes in many different organisms, genetic screens can provide valuable insight as to how those genes function.

Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.

<span class="mw-page-title-main">Functional genomics</span> Field of molecular biology

Functional genomics is a field of molecular biology that attempts to describe gene functions and interactions. Functional genomics make use of the vast data generated by genomic and transcriptomic projects. Functional genomics focuses on the dynamic aspects such as gene transcription, translation, regulation of gene expression and protein–protein interactions, as opposed to the static aspects of the genomic information such as DNA sequence or structures. A key characteristic of functional genomics studies is their genome-wide approach to these questions, generally involving high-throughput methods rather than a more traditional "candidate-gene" approach.

<span class="mw-page-title-main">Designer baby</span> Genetically modified human embryo

A designer baby is a baby whose genetic makeup has been selected or altered, often to exclude a particular gene or to remove genes associated with disease. This process usually involves analysing a wide range of human embryos to identify genes associated with particular diseases and characteristics, and selecting embryos that have the desired genetic makeup; a process known as preimplantation genetic diagnosis. Screening for single genes is commonly practiced, and polygenic screening is offered by a few companies. Other methods by which a baby's genetic information can be altered involve directly editing the genome before birth, which is not routinely performed and only one instance of this is known to have occurred as of 2019, where Chinese twins Lulu and Nana were edited as embryos, causing widespread criticism.

<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.

<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">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).

<span class="mw-page-title-main">Epigenome editing</span>

Epigenome editing or epigenome engineering is a type of genetic engineering in which the epigenome is modified at specific sites using engineered molecules targeted to those sites. Whereas gene editing involves changing the actual DNA sequence itself, epigenetic editing involves modifying and presenting DNA sequences to proteins and other DNA binding factors that influence DNA function. By "editing” epigenomic features in this manner, researchers can determine the exact biological role of an epigenetic modification at the site in question.

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.

CRISPR-Cas design tools are computer software platforms and bioinformatics tools used to facilitate the design of guide RNAs (gRNAs) for use with the CRISPR/Cas gene editing system.

<span class="mw-page-title-main">Cas12a</span> DNA-editing technology

Cas12a is a subtype of Cas12 proteins and an RNA-guided endonuclease that forms part of the CRISPR system in some bacteria and archaea. 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 use an RNA to target nucleic acid in a specific and programmable matter. In the organisms from which it originates, this guide RNA is a copy of a piece of foreign nucleic acid that previously infected the cell.

No-SCAR genome editing 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, 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.

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.

CRISPR-Display (CRISP-Disp) is a modification of the CRISPR/Cas9 system for genome editing. The CRISPR/Cas9 system uses a short guide RNA (sgRNA) sequence to direct a Streptococcus pyogenes Cas9 nuclease, acting as a programmable DNA binding protein, to cleave DNA at a site of interest.

CRISPR activation (CRISPRa) is a type of CRISPR tool that uses modified versions of CRISPR effectors without endonuclease activity, with added transcriptional activators on dCas9 or the guide RNAs (gRNAs).

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.

Prime editing is a 'search-and-replace' genome editing technology in molecular biology by which the genome of living organisms may be modified. The technology directly writes new genetic information into a targeted DNA site. It uses a fusion protein, consisting of a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA), capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides. It mediates targeted insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates.

References

  1. ↑ Brenner S (May 1974). "The genetics of Caenorhabditis elegans". Genetics. 77 (1): 71–94. doi:10.1093/genetics/77.1.71. PMC   1213120 . PMID   4366476.
  2. ↑ Gans M, Audit C, Masson M (December 1975). "Isolation and characterization of sex-linked female-sterile mutants in Drosophila melanogaster". Genetics. 81 (4): 683–704. doi:10.1093/genetics/81.4.683. PMC   1213428 . PMID   814037.
  3. ↑ Nüsslein-Volhard C, Wieschaus E (October 1980). "Mutations affecting segment number and polarity in Drosophila". Nature. 287 (5785): 795–801. Bibcode:1980Natur.287..795N. doi:10.1038/287795a0. PMID   6776413. S2CID   4337658.
  4. ↑ Haffter P, Granato M, Brand M, Mullins MC, Hammerschmidt M, Kane DA, et al. (December 1996). "The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio". Development. 123: 1–36. doi:10.1242/dev.123.1.1. PMID   9007226.
  5. ↑ Driever W, Solnica-Krezel L, Schier AF, Neuhauss SC, Malicki J, Stemple DL, et al. (December 1996). "A genetic screen for mutations affecting embryogenesis in zebrafish" (PDF). Development. 123: 37–46. doi:10.1242/dev.123.1.37. PMID   9007227.
  6. ↑ Nolan PM, Peters J, Strivens M, Rogers D, Hagan J, Spurr N, et al. (August 2000). "A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse". Nature Genetics. 25 (4): 440–3. doi:10.1038/78140. PMID   10932191. S2CID   9028853.
  7. ↑ Kasarskis A, Manova K, Anderson KV (June 1998). "A phenotype-based screen for embryonic lethal mutations in the mouse". Proceedings of the National Academy of Sciences of the United States of America. 95 (13): 7485–90. Bibcode:1998PNAS...95.7485K. doi: 10.1073/pnas.95.13.7485 . PMC   22659 . PMID   9636176.
  8. ↑ Gossler A, Doetschman T, Korn R, Serfling E, Kemler R (December 1986). "Transgenesis by means of blastocyst-derived embryonic stem cell lines". Proceedings of the National Academy of Sciences of the United States of America. 83 (23): 9065–9. Bibcode:1986PNAS...83.9065G. doi: 10.1073/pnas.83.23.9065 . PMC   387075 . PMID   3024164.
  9. ↑ Capecchi MR (June 1989). "Altering the genome by homologous recombination". Science. 244 (4910): 1288–92. Bibcode:1989Sci...244.1288C. doi:10.1126/science.2660260. PMID   2660260.
  10. ↑ Zaug AJ, Grosshans CA, Cech TR (December 1988). "Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes". Biochemistry. 27 (25): 8924–31. doi:10.1021/bi00425a008. PMID   3069131.
  11. ↑ Cameron FH, Jennings PA (December 1989). "Specific gene suppression by engineered ribozymes in monkey cells". Proceedings of the National Academy of Sciences of the United States of America. 86 (23): 9139–43. Bibcode:1989PNAS...86.9139C. doi: 10.1073/pnas.86.23.9139 . PMC   298449 . PMID   2556702.
  12. ↑ Ecker JR, Davis RW (August 1986). "Inhibition of gene expression in plant cells by expression of antisense RNA". Proceedings of the National Academy of Sciences of the United States of America. 83 (15): 5372–6. Bibcode:1986PNAS...83.5372E. doi: 10.1073/pnas.83.15.5372 . PMC   386288 . PMID   16593734.
  13. ↑ Toulmé JJ, Hélène C (December 1988). "Antimessenger oligodeoxyribonucleotides: an alternative to antisense RNA for artificial regulation of gene expression--a review". Gene. 72 (1–2): 51–8. doi:10.1016/0378-1119(88)90127-8. PMID   2468575.
  14. ↑ Chase D, Serafinas C, Ashcroft N, Kosinski M, Longo D, Ferris DK, Golden A (January 2000). "The polo-like kinase PLK-1 is required for nuclear envelope breakdown and the completion of meiosis in Caenorhabditis elegans". Genesis. 26 (1): 26–41. doi:10.1002/(sici)1526-968x(200001)26:1<26::aid-gene6>3.0.co;2-o. PMID   10660671. S2CID   21234791.
  15. ↑ Kawano T, Fujita M, Sakamoto H (July 2000). "Unique and redundant functions of SR proteins, a conserved family of splicing factors, in Caenorhabditis elegans development". Mechanisms of Development. 95 (1–2): 67–76. doi: 10.1016/s0925-4773(00)00339-7 . PMID   10906451. S2CID   17256368.
  16. ↑ Powers J, Bossinger O, Rose D, Strome S, Saxton W (October 1998). "A nematode kinesin required for cleavage furrow advancement". Current Biology. 8 (20): 1133–6. doi:10.1016/s0960-9822(98)70470-1. PMC   3209536 . PMID   9778533.
  17. ↑ Hsu JY, Sun ZW, Li X, Reuben M, Tatchell K, Bishop DK, et al. (August 2000). "Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes". Cell. 102 (3): 279–91. doi: 10.1016/s0092-8674(00)00034-9 . PMID   10975519. S2CID   16057773.
  18. ↑ Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (February 1998). "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans". Nature. 391 (6669): 806–11. Bibcode:1998Natur.391..806F. doi:10.1038/35888. PMID   9486653. S2CID   4355692.
  19. ↑ Agrawal N, Dasaradhi PV, Mohmmed A, Malhotra P, Bhatnagar RK, Mukherjee SK (December 2003). "RNA interference: biology, mechanism, and applications". Microbiology and Molecular Biology Reviews. 67 (4): 657–85. doi:10.1128/mmbr.67.4.657-685.2003. PMC   309050 . PMID   14665679.
  20. ↑ Sigoillot FD, Lyman S, Huckins JF, Adamson B, Chung E, Quattrochi B, King RW (February 2012). "A bioinformatics method identifies prominent off-targeted transcripts in RNAi screens". Nature Methods. 9 (4): 363–6. doi:10.1038/nmeth.1898. PMC   3482495 . PMID   22343343.
  21. ↑ Echeverri CJ, Beachy PA, Baum B, Boutros M, Buchholz F, Chanda SK, et al. (October 2006). "Minimizing the risk of reporting false positives in large-scale RNAi screens". Nature Methods. 3 (10): 777–9. doi:10.1038/nmeth1006-777. hdl: 1874/21016 . PMID   16990807. S2CID   1737581.
  22. ↑ Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. (March 2007). "CRISPR provides acquired resistance against viruses in prokaryotes". Science. 315 (5819): 1709–12. Bibcode:2007Sci...315.1709B. doi:10.1126/science.1138140. hdl: 20.500.11794/38902 . PMID   17379808. S2CID   3888761.
  23. 1 2 3 4 5 6 7 8 9 Yau EH, Rana TM (2018). "Next-Generation Sequencing of Genome-Wide CRISPR Screens". Next Generation Sequencing. Methods in Molecular Biology. Vol. 1712. pp. 203–216. doi:10.1007/978-1-4939-7514-3_13. ISBN   978-1-4939-7512-9. PMC   6089254 . PMID   29224076.
  24. 1 2 Boettcher M, McManus MT (May 2015). "Choosing the Right Tool for the Job: RNAi, TALEN, or CRISPR". Molecular Cell. 58 (4): 575–85. doi:10.1016/j.molcel.2015.04.028. PMC   4441801 . PMID   26000843.
  25. ↑ Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, et al. (October 2014). "Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation". Cell. 159 (3): 647–61. doi:10.1016/j.cell.2014.09.029. PMC   4253859 . PMID   25307932.
  26. ↑ Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. (February 2013). "Multiplex genome engineering using CRISPR/Cas systems". Science. 339 (6121): 819–23. Bibcode:2013Sci...339..819C. doi:10.1126/science.1231143. PMC   3795411 . PMID   23287718.
  27. ↑ Evers B, Jastrzebski K, Heijmans JP, Grernrum W, Beijersbergen RL, Bernards R (June 2016). "CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes". Nature Biotechnology. 34 (6): 631–3. doi:10.1038/nbt.3536. PMID   27111720. S2CID   22384060.
  28. ↑ Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (August 2012). "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity". Science. 337 (6096): 816–21. Bibcode:2012Sci...337..816J. doi:10.1126/science.1225829. PMC   6286148 . PMID   22745249.
  29. ↑ Wu X, Kriz AJ, Sharp PA (June 2014). "Target specificity of the CRISPR-Cas9 system". Quantitative Biology. 2 (2): 59–70. doi:10.1007/s40484-014-0030-x. PMC   4338555 . PMID   25722925.
  30. 1 2 Zhang F, Wen Y, Guo X (September 2014). "CRISPR/Cas9 for genome editing: progress, implications and challenges". Human Molecular Genetics. 23 (R1): R40-6. doi: 10.1093/hmg/ddu125 . PMID   24651067.
  31. 1 2 3 4 5 6 Joung J, Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham MD, et al. (April 2017). "Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening". Nature Protocols. 12 (4): 828–863. doi:10.1038/nprot.2017.016. PMC   5526071 . PMID   28333914.
  32. ↑ Endo M, Mikami M, Endo A, Kaya H, Itoh T, Nishimasu H, et al. (January 2019). "Genome editing in plants by engineered CRISPR-Cas9 recognizing NG PAM". Nature Plants. 5 (1): 14–17. doi:10.1038/s41477-018-0321-8. PMID   30531939. S2CID   54462288.
  33. 1 2 3 4 5 Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, et al. (February 2016). "Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9". Nature Biotechnology. 34 (2): 184–191. doi:10.1038/nbt.3437. PMC   4744125 . PMID   26780180.
  34. ↑ Cancellieri S, Canver MC, Bombieri N, Giugno R, Pinello L (November 2019). "CRISPRitz: rapid, high-throughput, and variant-aware in silico off-target site identification for CRISPR genome editing". Bioinformatics. 36 (7): 2001–2008. doi:10.1093/bioinformatics/btz867. PMC   7141852 . PMID   31764961.
  35. 1 2 3 4 5 6 Sanjana NE, Shalem O, Zhang F (August 2014). "Improved vectors and genome-wide libraries for CRISPR screening". Nature Methods. 11 (8): 783–784. doi:10.1038/nmeth.3047. PMC   4486245 . PMID   25075903.
  36. 1 2 "Pooled Libraries". Addgene.
  37. ↑ Xu CL, Ruan MZ, Mahajan VB, Tsang SH (January 2019). "Viral Delivery Systems for CRISPR". Viruses. 11 (1): 28. doi: 10.3390/v11010028 . PMC   6356701 . PMID   30621179.
  38. 1 2 Lino CA, Harper JC, Carney JP, Timlin JA (November 2018). "Delivering CRISPR: a review of the challenges and approaches". Drug Delivery. 25 (1): 1234–1257. doi:10.1080/10717544.2018.1474964. PMC   6058482 . PMID   29801422.
  39. ↑ Wang T, Wei JJ, Sabatini DM, Lander ES (January 2014). "Genetic screens in human cells using the CRISPR-Cas9 system". Science. 343 (6166): 80–4. Bibcode:2014Sci...343...80W. doi:10.1126/science.1246981. PMC   3972032 . PMID   24336569.
  40. ↑ Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, et al. (January 2014). "Genome-scale CRISPR-Cas9 knockout screening in human cells". Science. 343 (6166): 84–87. Bibcode:2014Sci...343...84S. doi:10.1126/science.1247005. PMC   4089965 . PMID   24336571.
  41. ↑ Yaniz-Galende E, Hajjar RJ (January 2014). "Stem cell and gene therapy for cardiac regeneration.". Cardiac Regeneration and Repair. Woodhead Publishing. pp. 347–379. doi:10.1533/9780857096708.4.347. ISBN   9780857096586.
  42. ↑ Pauwels K, Gijsbers R, Toelen J, Schambach A, Willard-Gallo K, Verheust C, et al. (December 2009). "State-of-the-art lentiviral vectors for research use: risk assessment and biosafety recommendations". Current Gene Therapy. 9 (6): 459–74. doi:10.2174/156652309790031120. PMID   20021330.
  43. 1 2 Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L (November 1998). "A third-generation lentivirus vector with a conditional packaging system". Journal of Virology. 72 (11): 8463–71. doi:10.1128/JVI.72.11.8463-8471.1998. PMC   110254 . PMID   9765382.
  44. 1 2 3 4 5 "Lenti-X CRISPR/Cas9 System User Manual" (PDF). Takara Bio USA.
  45. ↑ Tiscornia G, Singer O, Verma IM (2006). "Production and purification of lentiviral vectors". Nature Protocols. 1 (1): 241–5. doi:10.1038/nprot.2006.37. PMID   17406239. S2CID   37763028.
  46. 1 2 3 4 5 6 7 8 Agrotis A, Ketteler R (2015). "A new age in functional genomics using CRISPR/Cas9 in arrayed library screening". Frontiers in Genetics. 6: 300. doi: 10.3389/fgene.2015.00300 . PMC   4585242 . PMID   26442115.
  47. ↑ Yeung AT, Choi YH, Lee AH, Hale C, Ponstingl H, Pickard D, et al. (October 2019). "Salmonella Infection". mBio. 10 (5). doi:10.1128/mBio.02169-19. PMC   6786873 . PMID   31594818.
  48. 1 2 Hart T, Chandrashekhar M, Aregger M, Steinhart Z, Brown KR, MacLeod G, et al. (December 2015). "High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities". Cell. 163 (6): 1515–26. doi: 10.1016/j.cell.2015.11.015 . PMID   26627737.
  49. ↑ Slesarev A, Viswanathan L, Tang Y, Borgschulte T, Achtien K, Razafsky D, et al. (March 2019). "CRISPR/CAS9 targeted CAPTURE of mammalian genomic regions for characterization by NGS". Scientific Reports. 9 (1): 3587. Bibcode:2019NatSR...9.3587S. doi:10.1038/s41598-019-39667-4. PMC   6401131 . PMID   30837529.
  50. 1 2 Li W, Xu H, Xiao T, Cong L, Love MI, Zhang F, et al. (2014). "MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens". Genome Biology. 15 (12): 554. doi: 10.1186/s13059-014-0554-4 . PMC   4290824 . PMID   25476604.
  51. 1 2 3 Li W, Köster J, Xu H, Chen CH, Xiao T, Liu JS, et al. (December 2015). "Quality control, modeling, and visualization of CRISPR screens with MAGeCK-VISPR". Genome Biology. 16: 281. doi: 10.1186/s13059-015-0843-6 . PMC   4699372 . PMID   26673418.
  52. ↑ Sharma S, Petsalaki E (March 2018). "Application of CRISPR-Cas9 Based Genome-Wide Screening Approaches to Study Cellular Signalling Mechanisms". International Journal of Molecular Sciences. 19 (4): 933. doi: 10.3390/ijms19040933 . PMC   5979383 . PMID   29561791.
  53. ↑ Tzelepis K, Koike-Yusa H, De Braekeleer E, Li Y, Metzakopian E, Dovey OM, et al. (October 2016). "A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia". Cell Reports. 17 (4): 1193–1205. doi:10.1016/j.celrep.2016.09.079. PMC   5081405 . PMID   27760321.
  54. ↑ Chen L, Alexe G, Dharia NV, Ross L, Iniguez AB, Conway AS, et al. (January 2018). "CRISPR-Cas9 screen reveals a MYCN-amplified neuroblastoma dependency on EZH2". The Journal of Clinical Investigation. 128 (1): 446–462. doi:10.1172/JCI90793. PMC   5749506 . PMID   29202477.
  55. ↑ Wang T, Birsoy K, Hughes NW, Krupczak KM, Post Y, Wei JJ, et al. (November 2015). "Identification and characterization of essential genes in the human genome". Science. 350 (6264): 1096–101. Bibcode:2015Sci...350.1096W. doi:10.1126/science.aac7041. PMC   4662922 . PMID   26472758.
  56. ↑ Leary M, Heerboth S, Lapinska K, Sarkar S (December 2018). "Sensitization of Drug Resistant Cancer Cells: A Matter of Combination Therapy". Cancers. 10 (12): 483. doi: 10.3390/cancers10120483 . PMC   6315347 . PMID   30518036.
  57. ↑ Wang C, Wang G, Feng X, Shepherd P, Zhang J, Tang M, et al. (April 2019). "Genome-wide CRISPR screens reveal synthetic lethality of RNASEH2 deficiency and ATR inhibition". Oncogene. 38 (14): 2451–2463. doi:10.1038/s41388-018-0606-4. PMC   6450769 . PMID   30532030.
  58. 1 2 3 4 Hinze L, Pfirrmann M, Karim S, Degar J, McGuckin C, Vinjamur D, et al. (April 2019). "Synthetic Lethality of Wnt Pathway Activation and Asparaginase in Drug-Resistant Acute Leukemias". Cancer Cell. 35 (4): 664–676.e7. doi:10.1016/j.ccell.2019.03.004. PMC   6541931 . PMID   30991026.
  59. 1 2 Li B, Clohisey SM, Chia BS, Wang B, Cui A, Eisenhaure T, et al. (January 2020). "Genome-wide CRISPR screen identifies host dependency factors for influenza A virus infection". Nature Communications. 11 (1): 164. Bibcode:2020NatCo..11..164L. doi:10.1038/s41467-019-13965-x. PMC   6952391 . PMID   31919360.
  60. 1 2 Marceau CD, Puschnik AS, Majzoub K, Ooi YS, Brewer SM, Fuchs G, et al. (July 2016). "Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens". Nature. 535 (7610): 159–63. Bibcode:2016Natur.535..159M. doi:10.1038/nature18631. PMC   4964798 . PMID   27383987.
  61. ↑ Birsoy K, Wang T, Chen WW, Freinkman E, Abu-Remaileh M, Sabatini DM (July 2015). "An Essential Role of the Mitochondrial Electron Transport Chain in Cell Proliferation Is to Enable Aspartate Synthesis". Cell. 162 (3): 540–51. doi:10.1016/j.cell.2015.07.016. PMC   4522279 . PMID   26232224.
  62. ↑ Koike-Yusa H, Li Y, Tan EP, Velasco-Herrera M, Yusa K (March 2014). "Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library". Nature Biotechnology. 32 (3): 267–73. doi:10.1038/nbt.2800. PMID   24535568. S2CID   23071292.
  63. ↑ Chen S, Sanjana NE, Zheng K, Shalem O, Lee K, Shi X, et al. (March 2015). "Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis". Cell. 160 (6): 1246–60. doi:10.1016/j.cell.2015.02.038. PMC   4380877 . PMID   25748654.
  64. ↑ Shi J, Wang E, Milazzo JP, Wang Z, Kinney JB, Vakoc CR (June 2015). "Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains". Nature Biotechnology. 33 (6): 661–7. doi:10.1038/nbt.3235. PMC   4529991 . PMID   25961408.
  65. ↑ Ma H, Dang Y, Wu Y, Jia G, Anaya E, Zhang J, et al. (July 2015). "A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death". Cell Reports. 12 (4): 673–83. doi:10.1016/j.celrep.2015.06.049. PMC   4559080 . PMID   26190106.
  66. ↑ Parnas O, Jovanovic M, Eisenhaure TM, Herbst RH, Dixit A, Ye CJ, et al. (July 2015). "A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks". Cell. 162 (3): 675–86. doi:10.1016/j.cell.2015.06.059. PMC   4522370 . PMID   26189680.
  67. ↑ Schmid-Burgk JL, Chauhan D, Schmidt T, Ebert TS, Reinhardt J, Endl E, Hornung V (January 2016). "A Genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Screen Identifies NEK7 as an Essential Component of NLRP3 Inflammasome Activation". The Journal of Biological Chemistry. 291 (1): 103–9. doi: 10.1074/jbc.C115.700492 . PMC   4697147 . PMID   26553871.
  68. ↑ Quinn T. "Advantages and challenges of pooled libraries". Webinar series. Takara Bio USA.
  69. ↑ Fang Z, Weng C, Li H, Tao R, Mai W, Liu X, et al. (March 2019). "Single-Cell Heterogeneity Analysis and CRISPR Screen Identify Key β-Cell-Specific Disease Genes". Cell Reports. 26 (11): 3132–3144.e7. doi:10.1016/j.celrep.2019.02.043. PMC   6573026 . PMID   30865899.
  70. ↑ Vannucci L, Lai M, Chiuppesi F, Ceccherini-Nelli L, Pistello M (January 2013). "Viral vectors: a look back and ahead on gene transfer technology". The New Microbiologica. 36 (1): 1–22. PMID   23435812.
  71. 1 2 Lagziel S, GottliebE, Shlomi T (2020). "Mind your media". Nature Metabolism. 2 (12): 1369–1372. doi:10.1038/s42255-020-00299-y. PMID   33046912. S2CID   222319735.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  72. 1 2 Lagziel S, Lee WD, Shlomi T (2019). "Inferring cancer dependencies on metabolic genes from large-scale genetic screens". BMC Biol. 17 (1): 37. doi: 10.1186/s12915-019-0654-4 . PMC   6489231 . PMID   31039782.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  73. ↑ Vande Voorde J, Ackermann T, Pfetzer N, Sumpton D, Mackay G, Kalna G; et al. (2019). "Improving the metabolic fidelity of cancer models with a physiological cell culture medium". Sci Adv. 5 (1): eaau7314. Bibcode:2019SciA....5.7314V. doi:10.1126/sciadv.aau7314. PMC   6314821 . PMID   30613774.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  74. ↑ Cantor JR, Abu-Remaileh M, Kanarek N, Freinkman E, Gao X, Louissaint A; et al. (2017). "Physiologic Medium Rewires Cellular Metabolism and Reveals Uric Acid as an Endogenous Inhibitor of UMP Synthase". Cell. 169 (2): 258–272.e17. doi:10.1016/j.cell.2017.03.023. PMC   5421364 . PMID   28388410.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  75. ↑ Jaitin DA, Weiner A, Yofe I, Lara-Astiaso D, Keren-Shaul H, David E, et al. (December 2016). "Dissecting Immune Circuits by Linking CRISPR-Pooled Screens with Single-Cell RNA-Seq". Cell. 167 (7): 1883–1896.e15. doi: 10.1016/j.cell.2016.11.039 . PMID   27984734.
  76. ↑ Datlinger P, Rendeiro AF, Schmidl C, Krausgruber T, Traxler P, Klughammer J, et al. (March 2017). "Pooled CRISPR screening with single-cell transcriptome readout". Nature Methods. 14 (3): 297–301. doi:10.1038/nmeth.4177. PMC   5334791 . PMID   28099430.
  77. ↑ Dixit A, Parnas O, Li B, Chen J, Fulco CP, Jerby-Arnon L, et al. (December 2016). "Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens". Cell. 167 (7): 1853–1866.e17. doi:10.1016/j.cell.2016.11.038. PMC   5181115 . PMID   27984732.
  78. ↑ Alberts, Bruce (2014). Molecular Biology of the Cell (6th ed.). Garland Science.


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