CRISPR/Cas9 Finds Its Way As an Important Tool For Drug Discovery & Development
UPDATED 6/11/2021
CRISPR Diagnostics: CRISPR-dx Comes of Age: Tool in Drug Development
The past five years has seen a rapid expansion of the ability of CRISPR based tools toward diagnostic testing. Recently, CRISPR has been used to detect SARS-CoV-2 in patients. An article in the journal Science describes the different classes of CRISPR diagnostics in use today .
Update near end of post
UPDATED 8/08/2020
Association to Causation: Using GWAS to Identify Druggable Targets
A Gen Webinar Thursday, August 6, 2020; 11:00am – 12:30pm EST
See at end of post
Curator: Stephen J. Williams, Ph.D.
CRISPR/Cas9 Finds Its Way As an Important Tool For Drug Discovery & Development, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair
The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA.
CRISPR/Cas systems are part of the adaptive immune system of bacteria and archaea, protecting them against invading nucleic acids such as viruses by cleaving the foreign DNA in a sequence-dependent manner. Although CRISPR arrays were first identified in the Escherichia coli genome in 1987 (Ishino et al., 1987), their biological function was not understood until 2005, when it was shown that the spacers were homologous to viral and plasmid sequences suggesting a role in adaptive immunity (Bolotin et al., 2005; Mojica et al., 2005; Pourcel et al., 2005). Two years later, CRISPR arrays were confirmed to provide protection against invading viruses when combined with Cas genes (Barrangou et al., 2007). The mechanism of this immune system based on RNA-mediated DNA targeting was demonstrated shortly thereafter (Brouns et al., 2008; Deltcheva et al., 2011; Garneau et al., 2010; Marraffini and Sontheimer, 2008).
Jennifer Doudna, PhD Professor of Molecular and Cell Biology and Chemistry, University of California, Berkeley Investigator, Howard Hughes Medical Institute has recently received numerous awards and accolades for the discovery of CRISPR/Cas9 as a tool for mammalian genetic manipulation as well as her primary intended research target to understand bacterial resistance to viral infection.
A good post on the matter and Dr. Doudna can be seen below:
In Delineating a Role for CRISPR-Cas9 in Pharmaceutical Targeting inheritable metabolic disorders in which may benefit from a CRISPR-Cas9 mediated therapy is discussed. However this curation is meant to focus on CRISPR/CAS9 AS A TOOL IN PRECLINICAL DRUG DEVELOPMENT.
Three Areas of Importance of CRISPR/Cas9 as a TOOL in Preclinical Drug Discovery Include:
- Gene-Function Studies: CRISPR/CAS9 ability to DEFINE GENETIC LESION and INSERTION SITE
- CRISPR/CAS9 Use in Developing Models of Disease
- CRISPR/CAS9 Use as a Diagnostic Tool
- Using CRISPR/Cas9 in PRECLINICAL TOXICOLOGY STUDIES
I. Gene-Function Studies: CRISPR/CAS9 ability to DEFINE GENETIC LESION and INSERTION SITE
The advent of the first tools for manipulating genetic material (cloning, PCR, transgenic technology, and before microarray and other’omic methods) allowed scientists to probe novel, individual gene functions as well as their variants and mutants in a “one-gene-at-a time” process. In essence, a gene (or mutant gene) was sequenced, cloned into expression vectors and transfected into recipient cells where function was evaluated.
However, some of the experimental issues with this methodology involved
- Lack of knowledge of insertion site of the transgene – this leads to off-target effects usually due to insertion of a transgene in front of unwanted promoters or insertion at a site resulting in gene disruption or even mutagenesis. In an extreme case, such as transposon-induced mutagenesis may lead to transformation as described in an earlier post on this site How Mobile Elements in “Junk” DNA Promote Cancer – Part 1: Transposon-mediated Tumorigenesis
- Most transfections experiments result in NON ISOGENIC cell lines – by definition the insertion of a transgene alters the genetic makeup of a cell line. Simple transfection experiments with one transgene compared to a “null” transfectant compares non-isogenic lines, possibly confusing the interpretation of gene-function studies. Therefore a common technique is to develop cell lines with inducible gene expression, thereby allowing the investigator to compare a gene’s effect in ISOGENIC cell lines.
- Use of CRSPR in Highthrough-put Screening of Genetic Function
A very nice presentation and summary of CRSPR’s use in determining gene function in a high-throughput manner can be found below
www.rna.uzh.ch/events/journalclub/20140429JCCaihong.pdf
- Determining Off-target Effects of Gene Therapy Simplified with CRSPR
In GUIDE-seq: First genome-wide method of detecting off-target DNA breaks induced by CRISPR-Cas nucleases (from This Journal’s series on Live Meeting Coverage) at a 2014 Koch lecture
Shengdar Q Tsai and J Keith Joung describe
“
an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs. Application of GUIDE-seq to 13 RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or chromatin immunoprecipitation sequencing (ChIP-seq). GUIDE-seq also identified RGN-independent genomic breakpoint ‘hotspots’.
SOURCE http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3117.html
“
II. CRISPR/Cas9 Use in Developing Models of Disease
- Developing Animal Tumor Models
In a post this year I discussed a talk at the recent 2015 AACR National Meeting on a laboratories ability to use CRISPR gene editing in-vivo to produce a hepatocarcinoma using viral delivery. The post can be seen here: Notes from Opening Plenary Session – The Genome and Beyond from the 2015 AACR Meeting in Philadelphia PA; Sunday April 19, 2015
1) In this talk Dr. Tyler Jacks discussed his use of CRSPR to generate a mouse model of liver tumor in an immunocompetent mouse. Some notes from this talk are given below
- B) Engineering Cancer Genomes: Tyler Jacks, Ph.D.; Director, Koch Institute for Integrative Cancer Research
- Cancer GEM’s (genetically engineered mouse models of cancer) had moved from transgenics to defined oncogenes
- Observation that p53 -/- mice develop spontaneous tumors (lymphomas)
- then GEMs moved to Cre/Lox systems to generate mice with deletions however these tumor models require lots of animals, much time to create, expensive to keep;
- figured can use CRSPR/Cas9 as rapid, inexpensive way to generate engineered mice and tumor models
- he used CRSPR/Cas9 vectors targeting PTEN to introduce PTEN mutations in-vivo to hepatocytes; when they also introduced p53 mutations produced hemangiosarcomas; took ONLY THREE months to produce detectable tumors
- also produced liver tumors by using CRSPR/Cas9 to introduce gain of function mutation in β-catenin
See an article describing this study by MIT News “A New Way To Model Cancer: New gene-editing technique allows scientists to more rapidly study the role of mutations in tumor development.”
The original research article can be found in the August 6, 2014 issue of Nature[1]
And see also on the Jacks Lab site under Research
2) In the Upcoming Meeting New Frontiers in Gene Editing multiple uses of CRISPR technology is discussed in relation to gene knockout/function studies, tumor model development and
New Frontiers in Gene Editing
Session Spotlight:
BUILDING IN VIVO MODELS FOR DRUG DISCOVERY
Genome Editing Animal Models in Drug Discovery
Myung Shin, Ph.D., Senior Principal Scientist, Biology-Discovery, Genetics and Pharmacogenomics, Merck Research Laboratories
Recent advances in genome editing have greatly accelerated and expanded the ability to generate animal models. These tools allow generating mouse models in condensed timeline compared to that of conventional gene-targeting knock-out/knock-in strategies. Moreover, the genome editing methods have expanded the ability to generate animal models beyond mice. In this talk, we will discuss the application of ZFN and CRISPR to generate various animal models for drug discovery programs.
In vivo Cancer Modeling and Genetic Screening Using CRISPR/Cas9
Sidi Chen, Ph.D., Postdoctoral Fellow, Laboratories of Dr. Phillip A. Sharp and Dr. Feng Zhang, Koch Institute for Integrative Cancer Research at MIT and Broad Institute of Harvard and MIT
Here we describe a genome-wide CRISPR-Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library. The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late stage primary tumors were found to target a small set of genes, suggesting specific loss-of-function mutations drive tumor growth and metastasis.
FEATURED PRESENTATION: In vivo Chromosome Engineering Using CRISPR-Cas9
Andrea Ventura, M.D., Ph.D., Assistant Member, Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center
We will discuss our experience using somatic genome editing to engineer oncogenic chromosomal rearrangements in vivo. More specifically, we will present the results of our ongoing efforts aimed at modeling cancers driven by chromosomal rearrangements using viral mediated delivery of Crispr-Cas9 to adult animals.
RNAi and CRISPR/Cas9-Based in vivo Models for Drug Discovery
Christof Fellmann, Ph.D., Postdoctoral Fellow, Laboratory of Dr. Jennifer Doudna, Department of Molecular and Cell Biology, The University of California, Berkeley
Genetically engineered mouse models (GEMMs) are a powerful tool to study disease initiation, treatment response and relapse. By combining CRISPR/Cas9 and “Sensor” validated, tetracycline-regulated “miR-E” shRNA technology, we have developed a fast and scalable platform to generate RNAi GEMMs with reversible gene silencing capability. The synergy of CRISPR/Cas9 and RNAi enabled us to not only model disease pathogenesis, but also mimic drug therapy in mice, providing us capability to perform preclinical studies in vivo.
In vivo Genome Editing Using Staphylococcus aureus Cas9
Fei Ann Ran, Ph.D., Post-doctoral Fellow, Laboratory of Dr. Feng Zhang, Broad Institute and Junior Fellow, Harvard Society of Fellows
The RNA-guided Cas9 nuclease from the bacterial CRISPR/Cas system has been adapted as a powerful tool for facilitating targeted genome editing in eukaryotes. Recently, we have identified an additional small Cas9 nuclease from Staphylococcus aureus that can be packaged with its guide RNA into a single adeno-associated virus (AAV) vector for in vivo applications. We demonstrate the use of this system for effective gene modification in adult animals and further expand the Cas9 toolbox for in vivo genome editing.
OriGene, Making the Right Tools for CRISPR Research
Xuan Liu, Ph.D., Senior Director, Marketing, OriGene
CRISPR technology has quickly revolutionized the scientific community. Its simplicity has democratized the genome editing technology and enabled every lab to consider its utility in gene function research. As the largest tool box for gene functional research, OriGene created a large collection of CRISPR-related tools, including various all-in-one vectors for gRNA cloning, donor vector backbones, genome-wide knockout kits, AAVS1 insertion vectors, etc. OriGene’s high quality products will accelerate CRISPR research.
- Transgenic Animals : Custom Mouse and Rat Model Generation Service Using CRISPR/Cas9 by AppliedStem Cell Inc. (http://www.appliedstemcell.com/)
A critical component of producing transgenic animals is the ability of each successive generations to pass on the transgene. In her post on this site, A NEW ERA OF GENETIC MANIPULATION Dr. Demet Sag discusses the molecular biology of Cas9 systems and their efficiency to cause point mutations which can be passed on to subsequent generations
“
This group developed a new technology for editing genes that can be transferable change to the next generation by combining microbial immune defense mechanism, CRISPR/Cas9 that is the latest ground breaking technology for translational genomics with gene therapy-like approach.
- In short, this so-called “mutagenic chain reaction” (MCR) introduces a recessive mutation defined by CRISPR/Cas9 that lead into a high rate of transferable information to the next generation. They reported that when they crossed the female MCR offspring to wild type flies, the yellow phenotype observed more than 95 percent efficiency.
“
The advantage of CRISPR/Cas9 over ZFNs or TALENs is its scalability and multiplexibility in that multiple sites within the mammalian genome can be simultaneously modified, providing a robust, high-throughput approach for gene editing in mammalian cells.
Applied StemCell, Inc. offers various services related to animal models including conventional transgenic rats, and phenotype analysis using knock-in, knock-out strategies.
Further explanation of their use of CRSPR can be found at the site below:
http://pharmaceuticalintelligence.com/2014/10/29/gene-editing-at-crispr-speed-services-and-tools/
In addition, ReproCELL Inc., a Tokyo based stem cell company, uses CRSPR to develop
· Tailored disease model cells (hiPSC-Disease Model Cells)
- 2 types of services
- ReproUNUS™-g:human iPS cell derived functional cells involving gene editing by CRISPR/Cas9 system
- eproUNUS™-p:patient derived iPS cell derived functional cells
III. Using CRISPR/Cas9 in PRECLINICAL TOXICOLOGY STUDIES
As of now it is unclear as to the strategy of pharma in how to use this technology for toxicology testing however a few companies have licensed the technology to use across their R&D platforms including
A recent paper used a sister technique TALEN to generate knock-in pigs which suggest that it would be possible to generate pigs with human transgenes, especially in human liver isozymes in orer to study hepatotoxicity of drugs.
Efficient bi-allelic gene knockout and site-specific knock-in mediated by TALENs in pigs
Jing Yao, Jiaojiao Huang, Tang Hai, Xianlong Wang, Guosong Qin, Hongyong Zhang, Rong Wu, Chunwei Cao, Jianzhong Jeff Xi, Zengqiang Yuan, Jianguo Zhao
Sci Rep. 2014; 4: 6926. Published online 2014 November 5. doi: 10.1038/srep06926
UPDATED 8/08/2020
Association to Causation: Using GWAS to Identify Druggable Targets
A Gen Webinar Thursday, August 6⋅11:00am – 12:30pm
This webinar is available at https://www.genengnews.com/resources/webinars/association-to-causation-using-gwas-to-identify-druggable-target/
Speakers:
Martin Kampmann, PhD
Associate Professor
UCSF
Investigator
Chan Zuckerberg Biohub
Kevin Holden, PhD
Head of Science
Synthego
Abhi Saharia, PhD
VP, Commercial Development
Synthego
Human genetics provides perhaps the single best opportunity to innovate and improve clinical success rates, through the identification of novel drug targets for complex disease. Even as correlation identifies multiple genetic variants associated with disease, it is challenging to conduct requisite functional studies to identify the causal variants, especially since most association signals map to non-coding regions of the genome.
Genetic editing technologies, such as CRISPR, have enabled the modeling of associated variants at their native loci, including non-coding loci, empowering the identification of underlying biological mechanisms of disease with potential causal genes. However, genome editing is largely manual today severely limiting scale, and forcing the use of rational filters to prioritize which variants to investigate functionally.
In this GEN webinar, we will discuss several strategies enabling large-scale functional investigation of disease-associated variants in a cost- and time-effective manner, including different types of pooled CRISPR-based screens and the development of a fully automated genome engineering platform. We will also review how optimization of genome engineering on this platform enables the engineering of disease-associated variants at scale in pluripotent cells.
- They will be presenting on use of wide scale CRSPR screens to validate druggable targets
- The presenters will also discuss new platforms for these wide scale screens
- Multiple genetic variants associated with disease
- Big gap between accumulation of genetic variant information and functions of these variants
- CRSPRi or CRSPa (siRNA coupled or enhancer coupled CRSPR guides)
- Arrayed screens: multiplate guide RNAs and phenotype measured (phenotype can be morphology, complex biological systems like organoids or non autonomous functions
- Using pooled screens and use of suitable cell model critical for this strategy
- For example in iPSC vs. neurons has different expression patterns upon same CRSPR of UBA1
- Advantage is using CRSPR to take iPSC from diseased variant patient to make a corrected isogenic control then introduce gRNAs and use modifier screens to determine phenotypes
- Generated a platform called CRISPRbrain.org to do bioinformatics on various experiments with different guide RNAs (CRSPRs)
Abhi Saharia, PhD Syntheco
- Target identification with CSRSPR at Scale
- Nature medicine paper did GWAS and found 27 SNV associated with high risk disease and a rational filter focused on 1 SNV in noncoding region but why study a single variant and if studied all 27 would they have been able to identify a more representative druggable set?
- Goal is to reduce or eliminate these rational filters
- HALO (scalable RNA guide), ECLIPSE platform (automated generation of modified cell lines, BIOINFORMATIC platform (integrated informatics)
- Syntheco uses an electroporation with ribonucleic proteins (RNP) to give highest efficiency and minimizes off target as complex is only in cells for a short period of time
- They confirm they are doing single cell cloning by using automated microscopy to confirm single cell growth in each cloning well
Kevin Holden, Head of Science at Syntheco
- Engineering iPSc genetically modified cells at scale
- The closer you get to your target site the more efficient your CRSPR so a big factor when making guides, especially for knock-in CRSPR
- Adding a small molecule non homologous end joining inhibitor increases efficiency to 95%
- Cold shocking the cells also assists in homologous repair
- Use cleavage resistant templates
III. CRISPR/CAS9 AS A DIAGNOSTIC TOOL
In the journal Science, Omar Abudayyeh and Jonathan Gootenberg discuss how CRISPR-based diagnostic (CRISPR-dx) tools offer a solution, and multiple CRISPR-dx products for detection of the SARS-CoV-2 RNA genome have been authorized by the US Food and Drug Administration (FDA). In addition they discuss the work by Jiao et al. in combining this technique to develop a rapid and sensitive SARS-CoV2 diagnostic test.
Omar O. Abudayyeh, Jonathan S. Gootenberg. Science 28 May 2021: CRISPR Diagnostics
Vol. 372, Issue 6545, pp. 914-915; DOI: 10.1126/science.abi9335
Summary
Although clinical diagnostics take many forms, nucleic acid–based testing has become the gold standard for sensitive detection of many diseases, including pathogenic infections. Quantitative polymerase chain reaction (qPCR) has been widely adopted for its ability to detect only a few DNA or RNA molecules that can unambiguously specify a particular disease. However, the complexity of this technique restricts application to laboratory settings. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has underscored the need for the development and deployment of nucleic acid tests that are economical, easily scaled, and capable of being run in low-resource settings, without sacrifices in speed, sensitivity or specificity. CRISPR-based diagnostic (CRISPR-dx) tools offer a solution, and multiple CRISPR-dx products for detection of the SARS-CoV-2 RNA genome have been authorized by the US Food and Drug Administration (FDA). On page 941 of this issue, Jiao et al. (1) describe a new CRISPR-based tool to distinguish several SARS-CoV-2 variants in a single reaction.
Although clinical diagnostics take many forms, nucleic acid–based testing has become the gold standard for sensitive detection of many diseases, including pathogenic infections. Quantitative polymerase chain reaction (qPCR) has been widely adopted for its ability to detect only a few DNA or RNA molecules that can unambiguously specify a particular disease. However, the complexity of this technique restricts application to laboratory settings. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has underscored the need for the development and deployment of nucleic acid tests that are economical, easily scaled, and capable of being run in low-resource settings, without sacrifices in speed, sensitivity or specificity. CRISPR-based diagnostic (CRISPR-dx) tools offer a solution, and multiple CRISPR-dx products for detection of the SARS-CoV-2 RNA genome have been authorized by the US Food and Drug Administration (FDA). On page 941 of this issue, Jiao et al. (1) describe a new CRISPR-based tool to distinguish several SARS-CoV-2 variants in a single reaction.
There are multiple types of CRISPR systems comprising basic components of a single protein or protein complex, which cuts a specific DNA or RNA target programmed by a complementary guide sequence in a CRISPR-associated RNA (crRNA). The type V and VI systems and the CRISPR-associated endonucleases Cas12 (2, 3) and Cas13 (4, 5) bind and cut DNA or RNA, respectively. Furthermore, upon recognizing a target DNA or RNA sequence, Cas12 and Cas13 proteins exhibit “collateral activity” whereby any DNA or RNA, respectively, in the sample is cleaved regardless of its nucleic acid sequence (4, 6). Thus, reporter DNAs or RNAs, which allow for visual or fluorescent detection upon cleavage, can be added to a sample to infer the presence or absence of specific DNA or RNA species (4–8).
Initial versions of CRISPR-dx utilizing Cas13 alone were sensitive to the low picomolar range, corresponding to a limit of detection of millions of molecules in a microliter sample. To improve sensitivity, preamplification methods, such as recombinase polymerase amplification (RPA), PCR, loop-mediated isothermal amplification (LAMP), or nucleic acid sequence–based amplification (NASBA), can be used with Cas12 or Cas13 to enable a limit of detection down to a single molecule (8). This preamplification approach, applicable to both Cas12 and Cas13 (6, 7), enabled a suite of detection methods and multiplexing up to four orthogonal targets (7). Additional developments expanded CRISPR-dx readouts beyond fluorescence, including lateral flow (7), colorimetric (9), and electronic or material responsive readouts (10), allowing for instrument-free approaches. In addition, post–collateral-cleavage amplification methods, such as the use of the CRISPR-associated enzyme Csm6, have been combined with Cas13 to further increase the speed of CRISPR-dx tests (7). As an alternative to collateral-cleavage–based detection, type III CRISPR systems, which involve large multiprotein complexes capable of targeting both DNA and RNA, have been used for SARS-CoV-2 detection through production of colorimetric or fluorometric readouts (11).
FDA-authorized CRISPR-dx tests are currently only for use in centralized labs, because the most common CRISPR detection protocols require fluid handling steps and two different incubations, precluding their immediate use at the point of care. Single-step formulations have been developed to overcome this limitation, and these “one-pot” versions of CRISPR-dx are simple to run, operate at a single temperature, and run without complex equipment, producing either fluorescence or lateral flow readouts. The programmability of CRISPR makes new diagnostic tests easier to develop, and within months of the release of the SARS-CoV-2 genome, many COVID-19–specific CRISPR tests were reported and distributed around the world.
The broader capability for Cas enzyme–enhanced nucleic acid binding or cleavage has led to several other detection modalities. Cas9-based methods for cleaving nucleic acids in solution for diagnostic purposes have been combined with other detection platforms, such as destruction of undesired amplicons for preparation of next-generation sequencing libraries (12), or selective removal of alleles for nucleotide-specific detection (13). Alternatively, the programmable cleavage event from the Cas nuclease can be used to initiate an amplification reaction (14). Cas9-based DNA targeting has also been used for nucleotide detection in combination with solid-state electronics, promising an amplification-free platform for detection. In this platform, called CRISPR-Chip, the Cas9 protein binds nucleotide targets of interest (often in the context of the native genome) to graphene transistors, where the presence of these targets alters either current or voltage (15). By utilizing additional Cas9 orthologs and specific guide designs, CRISPR-Chip approaches have been tuned for single–base-pair sensitivity (15). Because they are integrated with electronic readers, CRISPR-Chip platforms may allow facile point-of-care detection with handheld devices.
GRAPHIC: ERIN DANIEL
Jiao et al. use a distinct characteristic of type II CRISPR systems, which involve Cas9, to develop a new type of noncollateral based CRISPR detection. Unlike Cas12s and Cas13, Cas9-crRNA complex formation requires an additional RNA known as the trans-activating CRISPR RNA (tracrRNA). By sequencing RNAs bound to Cas9 from Campylobacter jejuni in its natural host, the authors identified unexpected crRNAs, called noncanonical crRNA (ncrRNA), that corresponded to endogenous transcripts. Upon investigation of this surprising observation, it became clear that the tracrRNA was capable of hybridizing to semi-complementary sequences from a variety of RNA sources, leading to biogenesis of ncrRNAs of various sizes. Recognizing that they could program tracrRNAs to target a transcript of interest, the authors generated a reprogrammed tracrRNA (Rptr) that could bind and cleave a desired transcript, converting a piece of that transcript into a functional guide RNA. By then creating fluorescent DNA sensors that would be cleaved by the Rptr and ncrRNAs, the sensing of RNA by Cas9 could be linked to a detectable readout. This platform, called LEOPARD (leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection), can be combined with gel-based readouts and enables multiplexed detection of several different sequences in a single reaction (see the figure).
Jiao et al. also combined LEOPARD with PCR in a multistep workflow to detect SARS-CoV-2 genomes from patients with COVID-19. Although more work is needed to integrate this Cas9-based detection modality into a single step with RPA or LAMP to create a portable and sensitive isothermal test, an advantage of this approach is the higher-order multiplexing that can be achieved, allowing multiple pathogens, diseases, or variants to be detected simultaneously. More work is also needed to combine this technology with extraction-free methods for better ease of use; alternative readouts to gel-based readouts, such as lateral flow and colorimetric readouts, would be beneficial for point-of-care detection.
In just 5 years, the CRISPR-dx field has rapidly expanded, growing from a set of peculiar molecular biology discoveries to multiple FDA-authorized COVID-19 tests and spanning four of the six major subtypes of CRISPR systems. Despite the tremendous promise of CRISPR-dx, substantial challenges remain to adapting these technologies for point-of-care and at-home settings. Simplification of the chemistries to operate as a single reaction in a matter of minutes would be revolutionary, especially if the reaction could be run at room temperature without any complex or expensive equipment. These improvements to CRISPR-dx assays can be achieved by identification or engineering of additional Cas enzymes with lower-temperature requirements, higher sensitivity, or faster kinetics, enabling rapid and simple amplification-free detection with single-molecule sensitivity.
Often overlooked is the necessity for a sample DNA or RNA preparation step that is simple enough to be added directly to the CRISPR reaction to maintain a simple workflow for point-of-care testing. In addition, higher-order multiplexing developments would allow for expansive testing menus and approach the possibility of testing for all known diseases. As these advancements are realized, innovative uses of CRISPR-dx will continue in areas such as surveillance, integration with biomaterials, and environmental monitoring. In future years, CRISPR-dx assays may become universal in the clinic and at home, reshaping how diseases are diagnosed.
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