Molecular Biology

CRISPR-Cas & Lateral Flow

A Gene Editing Tool for Innov­ative Diagnostics

In 2020, Jennifer Doudna and Emmanuelle Charp­entier received the Nobel Prize in chemistry for their scien­tific contri­bu­tions to the simple and precise genome editing tool CRISPR-​​Cas. This has revolu­tionized biology within a decade as the most powerful tool for directed genetic manip­u­lation. Together with the „CRISPR-​​pioneer“ Feng Zhang, the two women demon­strated the power of basic research. The molecular gene scissor is more topical than ever and promise great oppor­tu­nities in areas such as medical therapy or plant breeding. Since then, the CRISPR-​​Cas toolbox has been contin­u­ously expanded and new appli­cation options presented. Therefore, it is not surprising that the CRISPR-​​Cas technology has found its way into molecular diagnostics. In combi­nation with DNA ampli­fi­cation, this method offers impressive accuracy and great sensi­tivity, which is partic­u­larly attractive in the sector of Point-​​of-​​Care (POC) diagnostics.

CRISPR-​​Cas-​​based methods for simple, ultra­sen­sitive and precise detection of nucleic acids have the potential to revolu­tionize the field of POC diagnostics.”

History of CRISPR-​​Cas as Diagnostic Tools

In 2017 and 2018, Jonathan Gootenberg and Omar Abudayyeh (Broad Institute, Boston) showed that the gene editing tool “CRISPR-​​Cas” is also suitable for diagnostic purposes. They could demon­strate that the impressive speci­ficity of the CRISPR system paired with the sensi­tivity of isothermal ampli­fi­cation techniques allows extremely precise and sensitive detection of nucleic acids. They called their innov­ative detection platform SHERLOCK, which stands for “Specific High-​​sensitivity Enzymatic Reporter unLOCKing”. In April 2018, two important publi­ca­tions showed that the SHERLOCK system can be combined with a simple, universal lateral flow device, the Milenia HybriDetect. This next logical step illus­trates the potential of this technology as a simple, portable platform for appli­ca­tions outside of specialized labora­tories. Almost at the same time, Janice Chen and colleagues from the working group around Nobel Prize winner Jennifer Doudna presented a SHERLOCK equiv­alent called DETECTR (“DNA-​​Endonuclease-​​Targeted CRISPR Trans Reporter”), which was based on the Cas12a protein and signif­i­cantly simplified the overall reaction. The rapid pace of devel­opment of these methods was accel­erated even more dramat­i­cally with the beginning of the pandemic. This short but impressive story is one reason why CRISPR-​​Cas-​​based methods have developed to one of the most exciting tools for POC-​​diagnostics.

Timeline: CRISPR-​​Cas as a diagnostic tool

2017

Landmark Paper I

Gootenberg & Abudayyeh „Cas13a as diagnostic tool“ – SHERLOCK technology

2018

Landmark Paper II

Chen et al. presenting DETECTR, simplified SHERLOCK equiv­alent using Cas12a

2018

Landmark Paper III

Mhyrvold and Freije et al. presenting field-​​applicable SHERLOCK system using a HybriDetect

2019

SARS-​​CoV-​​2 Pandemic

Start of the pandemic initiated massive boost of innov­ative molecular diagsnostics – Including CRISPR-​​Cas as diagnostic tools

2020

More Techniques

methods using Cas9 and Lateral Flow described: CASLFA & FELUDA

2020

Startups

Innov­ative Companies using CRISPR-​​Cas for diagnostics incresingly get into focus (Sherlock Biosciences, Mammoth Biosciences, CASPR Biotech)

2020

1st FDA Approval

The Sherlock™ CRISPR SARS-​​CoV-​​2 kit receives FDA Emergency Use Autho­rization

2017

Landmark Paper I

Gootenberg & Abudayyeh „Cas13a as diagnostic tool“ – SHERLOCK technology

2018

Landmark Paper III

Mhyrvold and Freije et al. presenting field-​​applicable SHERLOCK system using a HybriDetect

2020

More Techniques

methods using Cas9 and Lateral Flow described: CASLFA & FELUDA

2020

1st FDA Approval

The Sherlock™ CRISPR SARS-​​CoV-​​2 kit receives FDA Emergency Use Autho­rization

2018

Landmark Paper II

Chen et al. presenting DETECTR, simplified SHERLOCK equiv­alent using Cas12a

2019

SARS-​​CoV-​​2 Pandemic

Start of the pandemic initiated massive boost of innov­ative molecular diagsnostics – Including CRISPR-​​Cas as diagnostic tools

2020

Startups

Innov­ative Companies using CRISPR-​​Cas for diagnostics incresingly get into focus (Sherlock Biosciences, Mammoth Biosciences, CASPR Biotech)

An important method­ological trend of the last decade is the combi­nation of DNA ampli­fi­cation and lateral flow based readout. The analysis of more than 300 scien­tific publi­ca­tions revealed that the CRISPR-​​Cas toolbox became one the most relevant topics for innov­ative, molecular POC-​​diagnostics.

General Mechanism of CRISPR-​​Cas based Methods

An Expanding Toolbox and Two General Detection Strategies

CRISPR-Cas and Lateral Flow: Workflow and 2 general detection strategiesInter­est­ingly, special CRISPR-​​associated proteins (Cas proteins) with diagnos­ti­cally relevant charac­ter­istcs were used for methods like SHERLOCK or DETECTR. The CRISPR-​​Cas-​​complexes are able to “recognize” amplicons with an excellent speci­ficity. This in turn leads to the activation of a collateral nucelase activity of the Cas-​​Protein. As a result, terminal labeled reporter molecules (Cas12a: ssDNA; Cas13a: ssRNA) are efficiently cleaved by the Cas-​​protein. The seper­ation of terminal labels is detectable with the Milenia HybriDetect. This mechanism can be under­stood as an additional enhancer reaction beside DNA ampli­fi­cation. Based on this, numerous method­ological variants have been developed, but the general detection mechanism is ultimately based on the cleavage of a reporter. Therefore, we define this general detection mechanism as „Reporter Degra­dation Strategy“ or „Label Separation Strategy“.

 

Detection Principle of CRISPR-Cas dependent Assay using the Label Incorporation Strategy
Detection Principle of CRISPR-​​Cas dependent Assay using the Label Incor­po­ration Strategy

Since 2020, an increasing number of papers have been published that use the CRISPR-​​Cas-​​toolbox but do not follow the „Reporter Degra­dation Strategy“. The Cas9 protein, may be the best-​​known repre­sen­tative of the Cas protein family, moves into the focus of these methods. The complex of a chimeric guide RNA and Cas9 enables the specific “recog­nition” of the amplicon. This amplicon-​​CRISPR-​​Cas9 complex is detectable via Lateral Flow using the Milenia HybriDetect. Therefore, we define this method­ological approach as “Label Incor­po­ration Strategy“.

 

Detection Principle of CRISPR-Cas dependent Assay using the Label Incorporation Strategy
Detection Principle of CRISPR-​​Cas dependent Assay using the Label Incor­po­ration Strategy

Both general detection strategies allow a simple, fast readout through lateral flow analysis. However, the use and inter­pre­tation of the universal Lateral Flow Device has to be adapted to the used method.

 

How to modify a CRISPR/​Cas-​​based assay for Lateral Flow Readout

The adaptation of your own assay depends very much on the selected test method. If a detection system such as SHERLOCK or DETECTR is used in accor­dance with the „Label Separation Strategy“, the dual labeled reporter molecule is the most important component that needs to be questioned. The choice of the optimal reporter sequence depends on the CRISPR-​​Cas system used. Ultimately, it is important to determine the exact reporter concen­tration which works for the nuclease reaction and for the lateral flow analysis. The following table gives a brief overview of already described reporters for Cas13a and Cas12a dependent test formats.

 

Overview of frequently used Reporters
Cas ProteinReporter Type5′ Label3′ LabelSequence (5′ > 3′)
LwaCas13assRNA6-​​FAMBiotinpoly-​​U (≥ 10)
LwaCas13assRNA6-​​FAMBiotin(RU)14
LwaCas13assRNA6-​​FAMBiotin(MARARUGRGRCMA)2
LbCas12assDNA6-​​FAMBiotinTTATT
LbCas12assDNA6-​​FAMBiotinTTATTATT
LbCas12assDNA6-​​FAMBiotin(TTATT)3

If the detection is based on the recog­nition of the amplicon-​​CRISPR-​​Cas-​​Complex, various labeling strategies can be followed. In this scenario, guide RNA, Cas protein and amplicon usually come together to form a detectable complex including the Labels biotin and FITC/​FAM. The following figure describes three possible labeling strategies that have already been success­fully published.

CRISPR-Cas 9 - Lateral Flow Detection Strategies
Three different strategies combining CRISPR-​​Cas 9 amplicon binding & detection via Lateral Flow

Consid­ering the fact that CRISPR-​​Cas, as a diagnostic tool, was combined for the first time with a simple lateral flow readout in 2018, the actual method­ological variety is absolutely impressive. Only 3 years later there are numerous modifi­ca­tions and the toolbox is constantly expanding. Some important techniques that were described in the past are listed in the table below.

Overview — Methods combining CRISPR-​​Cas and Lateral Flow
Method Name (Acronym)General Detection StrategyAmpli­fi­cation MethodCas Protein (Origin)Labeling StrategyLateral Flow DeviceReference
FELUDAAmplicon Binding AssayPCRFnCas9 (Francisella novicida)Amplicon (Biotin), gRNA (FAM)Milenia HybriDetectAzhar et al., 2020
SHERLOCKReporter Degra­dation AssayLAMP, RPA (and in vitro transcription)e.g. LwaCas13a (Leptotrichia wadei)Collateral Cleavage of FAM-​​Biotin-​​ReporterMilenia HybriDetectKellner et al., 2020
CASLFAAmplicon Binding AssayPCR, RPACas9 (and dCas9, origin unkown)Probe-​​functionalized Nanopar­ticles, Amplicon (Biotin)Selfmade LFDWang et al., 2020
DETECTRReporter Degra­dation AssayLAMP, RPAe.g. LbCas12a (Lachnospiraceae bacterium)Collateral Cleavage of FAM-​​Biotin-​​ReporterMilenia HybriDetectBroughton et al., 2020
VIGILANTAmplicon Binding AssayRPAVirD2*-SpdCas9** fusion protein (*Agrobac­terium tumefa­ciens, **Strep­to­coccus pyogenes)VirD2-​​dCas9 (FAM), Amplicon (Biotin)Milenia HybriDetectMarsic et al., 2021
iSCANReporter Degra­dation AssayLAMPLbCas12a (Lachnospiraceae bacterium)Collateral Cleavage of FAM-​​Biotin-​​ReporterMilenia HybriDetectAli et al., 2020
“Biotin-​​dCas9-​​LFA”Amplicon Binding AssayRPASpdCas9* (Strep­to­coccus pyogenes, *3xFLAG + Biotin)SpdCas9 (Biotin), Amplicon (FITC)Milenia HybriDetectOsborn et al., 2021

Pros & Cons using CRISPR-​​Cas

Speci­ficity /​ Selec­tivity

One of the most important advan­tages of CRISPR-​​Cas-​​based nucleic acid detection is the impressive selec­tivity. By prese­lecting a suitable gRNA /​ crRNA, it is possible to discrim­inate between sequences with only one differing base pair. This speci­ficity down to single base level could be shown for the SHERLOCK (-like) appli­ca­tions as well as for CRISPR-​​Cas9-​​dependent amplicon binding assays.

Sensi­tivity

Especially Cas proteins with collateral nuclease activity (Cas12, Cas13, Cas14, Cas3, etc.) can signif­i­cantly contribute to an increase in detection sensi­tivity. The combi­nation of a very sensitive DNA ampli­fi­cation technique with a CRISPR-​​Cas-​​induced reporter degra­dation can be seen as two consec­utive enhancer reactions. This fact allows the very precise detection of DNA or RNA, even at attomolar concen­tra­tions. Current publi­ca­tions indicate that Cas protein modifi­cation can further improve the test perfor­mance.

Assay-​​Complexity

Most CRISPR-​​Cas-​​based systems for the detection of nucleic acids are based on the combi­nation of DNA ampli­fi­cation and amplicon recog­nition by the effector complex. This point can certainly be discussed criti­cally. In order to develop a very sensitive test, it is essential to establish a pre-​​amplification reaction. Many POC-​​relevant questions can already be answered with “simple” DNA ampli­fi­cation methods combined with Lateral Flow. The use of CRISPR-​​Cas compo­nents can complicate assay devel­opment signif­i­cantly. For this reason, it should be clearly defined whether the require­ments for the test justify the use of this platform before the devel­opment starts.

Multi­plexing

At this point, a clear distinction have to be made between the two CRISPR-​​dependent detection strategies described. „Progam­mable“ Cas proteins with collateral nuclease activity, such as Cas12 or Cas13, can signif­i­cantly improve the sensi­tivity of a nucleic acid detection assay. Never­theless, reporter degra­dation is indis­crim­inate, which excludes multi­plexing using a single Cas protein. A certain degree of multi­plexing can be achieved by using different Cas proteins with deviating „cutting“ prefer­ences. However, this compli­cates the assay devel­opment consid­erably. In contrast, the use of the CRISPR-​​Cas9 amplicon binding concept certainly allows multi­plexing. Especially, the VIGILANT method needs to be highlighted. Direct labeling of the Cas9-​​VirD2 fusion protein can massively reduce false positive results due to undesired inter­action of labeled nucleic acids. Furthermore, this system is perfectly compatible with the Milenia HybriDetect 2T, a universal Lateral Flow Device allowing simul­tanious detection of 2 target.

Learn more about (CRISPR/​Cas) Multi­plexing & Lateral Flow

Method Comparison Table
Method combined with LFASensi­tivitySpeci­ficitySpeedreverse Transcription (one pot)RobustnessMethod. VarietySimple Assay DesignMulti­plexing AbilityPrevention Carryover Cont.
PCR
LAMP
RPA
CRISPR (Label-​​Sep.)
CRISPR (Label-​​Inc.)