This work was financially supported by the National Key R &#38; D Program of China (2021YFD1400805), the Major Science and Technology R &#38; D Program of Jiangxi Province (20194ABC28007), Projects of Jiangxi Education Department (GJJ201449), and the Collaborative Innovation of Modern Agricultural Scientific Research in Jiangxi Province (JXXTCX2015002(3+2)-003).

1. Construction of the CLas-DETECTR
NOTE: The construction of CLas-DETECTR is a four-step process: solution preparation, citrus total DNA isolation, isothermal DNA amplification, and result visualization. The schematic of the CLas-DETECTR assay is illustrated in Figure 1A.
<ol>
	<li>Solution preparation
	<ol>
		<li>Prepare buffer A: 20 mM NaOH in 6% PEG 200. For 100 mL, add 113 mg of NaOH and 6 g of PEG 200 into 80 mL of H2O in a bottle. Incubate the bottle in a water bath at 60 &#176;C until all the PEG 200 is dissolved. Then, add H2O to a volume of 100 mL.</li>
		<li>Prepare solution B: For each reaction, add 10 &#956;L of RPA buffer, 0.8 &#956;L of F-RPA-RNRf, 0.8 &#956;L of R-RPA-RNRr, and 3.9 &#956;L of ddH2O to a final volume of 15.5 &#956;L. 
		NOTE: F-RPA-RNRf and R-RPA-RNRr are the primers used in the assay against the nrdB gene. See Table 1 for the sequence.</li>
		<li>Prepare solution C: For each reaction, add 1 &#956;L of ssDNA reporter, 3 &#956;L of NEB buffer 3.1, 1 &#956;L of crRNA, 4 &#956;L of Cas12a, and 11 &#956;L of ddH2O to a final volume of 20 &#956;L. 
		NOTE: If there are many samples to detect, make a large volume of solution B and solution C, and aliquot later.</li>
	</ol>
	</li>
	<li>Citrus total DNA isolation 
	NOTE: To save time and make this method suitable for field CLas detection, the alkaline polyethylene glycol (PEG)-based approach was used to obtain crude plant extracts for DNA amplification22,23
	<ol>
		<li>Citrus leaves were used in this protocol. First, punch five leaf discs from a leaf. Next, put the leaf discs into a 1.5 mL microcentrifuge tube, and add 200 &#956;L of buffer A. 
		NOTE: In the field, clean the leaves first if there is dust covering them. The leaf discs can be clipped using the lid of the 1.5 mL tube to avoid cross-contamination. Other citrus tissues, such as roots, stems, fruits, and flowers, can also be used in this protocol.</li>
		<li>Grind the leaf disks manually until smooth with a plastic rod.</li>
		<li>Leave the tube undisturbed for 10 min. Then, use the supernatant for DNA amplification. 
		NOTE: The assay can be paused here, and the crude plant extracts that were extracted by the alkaline-PEG method can be stored at &#8722;20 &#176;C for at least 1 year. The Newhall sweet orange (Citrus sinensis Osbeck var. Newhall) trees shown in the video were grown in a pot filled with a mix of 9/3/1 (v/v/v) peat soil/vermiculite/perlite and maintained in a glasshouse located in the campus of Gannan Normal University, Jiangxi, China. The HLB-infected trees were inoculated by grafting with CLas-positive Newhall buds. The HLB-infected and HLB-uninfected trees were confirmed by PCR. CLas was detected using the primer pair (F-RPA-RNRf/R-RPA-RNRr) targeting the CLas-specific marker gene nrdB. On the other side, characteristic HLB symptoms, blotchy mottle, and yellowing leaves can be found on CLas-positive but not on CLas-negative trees.</li>
	</ol>
	</li>
	<li>Isothermal DNA amplification
	<ol>
		<li>Add 1 &#956;L of the supernatant from step 1.2.3 and 1 &#956;L of MgOAc (14 mM final concentration) into solution B and mix well.</li>
		<li>In the lab, incubate them in a simple incubator at 37 &#176;C for 15 min. 
		NOTE: In the field, hold the tubes in hand for 15 min. It is also suggested to incubate tubes in a simple incubator at 37 &#176;C for 15 min if the conditions allow.</li>
	</ol>
	</li>
	<li>Results visualization
	<ol>
		<li>Add 10 &#956;L of the mixture from step 1.3.2 into solution C and mix well.</li>
		<li>Incubate them in a 37 &#176;C incubator for 60 min.</li>
		<li>Wear goggles and observe the green fluorescence signal released from the ssDNA reporter labeled with 5&#8217; 6-Fluorescein at 5&#8217; and the fluorescence quencher at 3&#8217; through&#160;a handheld fluorescent detection device (excitation wavelength: 440 nm, emission wavelength: 500 nm). 
		NOTE:green fluorescence signal can last several days, it is better to 
		CAUTION: The light emitted by the fluorescence detection device is harmful to the eyes. Make sure to wear goggles before observing the results.</li>
	</ol>
	</li>
</ol>
2. Specificity test
NOTE: To test the specificity of CLas-DETECTR, the rhizosphere bacterium Agrobacterium tumefaciens GV3101, Xanthomonas citri subsp. citri (Xcc), and Burkholderia stabilis strain 1440 isolated in the lab24 were subjected to the CLas-DETECTR test.&#160;
NOTE: Candidatus Liberibacter (CLas) cannot be cultured. One can only obtain CLas DNA from the extraction of genomic DNA from citrus tissues infected with CLas. Xcc is the causal agent of another important citrus disease, citrus canker. Burkholderia stabilis strain 1440 is an anti-Xcc bacterium isolated in the lab. Therefore, pure strains for all these three bacteria were used
<ol>
	<li>Extract the bacterial genomic DNA (gDNA) using a bacterial genomic DNA extraction kit following the manufacturer&#8217;s protocol. Use water as a negative control.</li>
	<li>Perform PCR using 0.2 mL PCR tube strips with optical caps&#160;in a 25 &#956;L reaction mixture containing 1 &#956;L of F-RPA-RNRf, 1 &#956;L of R-RPA-RNRr, 12.5 &#956;L of Ex Taq Version 2.0 plus dye, 1 &#956;L of DNA template, and 9.5 &#956;L of H2O.
	<ol>
		<li>Use the following thermal cycling reaction conditions: 1 min at 98 &#176;C; followed by 35 cycles of 10 s at 98 &#176;C, 30 s at 55 &#176;C, 15 s at 72 &#176;C; then 10 min at 72 &#176;C; and hold at 16 &#176;C.</li>
		<li>Run the PCR products on 1% agarose gel at 120 V for 15 min.</li>
	</ol>
	</li>
	<li>Perform qPCR in a 20 &#956;L reaction mixture containing 0.8 &#956;L of F-RPA-RNRf, 0.8 &#956;L of R-RPA-RNRr, 10 &#956;L of 2x TB Green Premix Ex Taq II (Tli RNaseH Plus), 1 &#956;L of the DNA template, and 7.4 &#956;L of H2O.
	<ol>
		<li>Use the following thermal cycling reaction conditions: 30 s at 95 &#176;C; followed by 45 cycles of 5 s at 95 &#176;C and 32 s at 60 &#176;C; and then a melt curve stage of 15 s at 95 &#176;C, 1 min at 60 &#176;C, and 15 s at 95 &#176;C.</li>
		<li>Include three technical repeats in each repeat.</li>
	</ol>
	</li>
	<li>Perform the CLas-DETECTR method following steps 1.1&#8211;1.4.</li>
</ol>
3. Sensitivity comparison
<ol>
	<li>To test the sensitivity of CLas DETECTR, detect a series of CLas DNA fragment dilutions using PCR, CLas-DETECTR, and qPCR, as described above.</li>
	<li>Amplify a DNA segment with a length of 1,060 bp from nrdB using the primer set F-RNR and R-RNR in a 100 &#956;L reaction mixture containing 4 &#956;L of F-RNR, 4 &#956;L of R-RNR, 50 &#956;L of PrimeSTAR Max Premix, 2 &#956;L of DNA template, and 40 &#956;L of H2O, following the thermal cycling reaction conditions (30 s at 98 &#176;C; followed by 35 cycles of 10 s at 98 &#176;C, 15 s at 55 &#176;C, 30 s at 72 &#176;C; then 10 min at 72 &#176;C; and hold at 16 &#176;C). 
	NOTE: The nrdB (ribonucleotide reductase &#946;- subunit) gene is specific to CLas21. One can easily obtain the nrdB amplicon from CLas-positive citrus gDNA using the primer set F-RNR and R-RNR. The primer set F-RNR and R-RNR amplifies a 1,060 bp nrdB fragment, while the primer set F-RPA-RNR and R-RPA-RNR amplifies a 104 bp fragment of the nrdB gene, which is the part of the 1,060 bp nrdB fragment.</li>
	<li>Purify the PCR products with a DNA gel extraction kit following the manufacturer&#8217;s manual.</li>
	<li>Dilute the PCR products containing the nrdB amplicons with sterilized ddH2O.</li>
	<li>Calculate the copy number of the nrdB gene using commercially available online DNA copy number and dilution calculator.</li>
	<li>Prepare DNA dilutions containing 2.01 &#215; 106 copies/&#956;L, 2.01 &#215; 105 copies/&#956;L, 2.01 &#215; 104 copies/&#956;L, 2.01 &#215; 103 copies/&#956;L, 2.01 &#215; 102 copies/&#956;L, 2.01 &#215; 101 copies/&#956;L, 2.01 &#215; 100 copies/&#956;L, and 2.01 &#215; 10&#8722;1 copies/&#956;L of CLas DNA fragment.</li>
</ol>
4. Sample detection
NOTE: After the specificity and sensitivity test, the CLas-DETECTR method was used to detect the presence of CLas in the field leaf samples collected from Newhall sweet orange trees grown in the germplasm resource nursery on the campus of Gannan Normal University, Jiangxi, China. A qPCR was performed to verify the results.
<ol>
	<li>Test a total of 15 trees. Use water as a negative control.</li>
	<li>Perform the CLas-DETECTR and qPCR methods as described above. 
	NOTE: Due to the uneven distribution characteristics of CLas in citrus, leaves showing HLB symptoms were collected as a priority. If a tree looked healthy, the leaves were collected randomlyCLas-DETECTR system works correctly.</li>
</ol>

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The authors declare that they have no competing interests.

This study presents a rapid and portable method to detect CLas named CLas-DETECTR, which combines the RPA and CRISPR-Cas12a systems. The workflow is illustrated in Figure 1. CLas-DETECTR detects CLas with specificity and sensitivity (Figure 2 and Figure 3). Furthermore, using Newhall leaf samples, CLas-DETECTR detects CLas with the same sensitivity as qPCR (Figure 4). Notably, the detec...

Huanglongbing (HLB) is one of the most problematic citrus diseases worldwide1. HLB is caused by the phloem-colonizing and fastidious bacteria Candidatus Liberibacter spp., including Candidatus Liberibacter asiaticus (CLas), Ca. L. africanus, and Ca. L. americanus2. The most prevalent HLB-associated species in China and the USA is CLas, which is transmitted by Asian citrus psyllids (Diaphorina citri) or through grafting3. After being infected by CLas, citrus trees demonstrate growth decline until death2. The common symptoms of citrus leaves infected with CLas are blotchy mottle, green islands (small circular dark green dots), raised corky veins on thicker and leathery leaves, and nonuniform yellowing shoots2. In addition, fruits infected with CLas appear small and lopsided2.
Since no citrus variety is resistant to HLB and there is no therapeutic cure for HLB, the prevention of HLB requires the quarantine and isolation of CLas-positive citrus trees2,3. Therefore, early detection is critical for monitoring and quarantine to prevent the spread of CLas and minimize economic losses3. In addition, sensitive CLas detection is needed due to the low titer of CLas in plants during the early stage of infection3. In China, CLas detection is usually conducted by certain certified test centers. However, the detection process usually takes at least 1 week, and the detection fee is expensive.&#160;Therefore, to help monitor the HLB incidence
Various technologies have been applied to diagnose HLB4,5,6,7,8,9. Polymerase chain reaction (PCR) and quantitative PCR (qPCR) are the most used tools for CLas detection due to their high sensitivity and specificity4,5. However, those technologies rely heavily on expensive instruments and highly skilled personnel. In addition, several isothermal amplification methods, such as loop-mediated isothermal amplification (LAMP), have been developed as attractive alternatives to conventional PCR methods due to their simplicity, rapidity, and low cost8,9,10. However, it is challenging to apply them to accurately detect CLas due to the non-specific amplification signals, which may cause false-positive results.
RNA-guided CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) endonuclease-based nucleic acid detection has been developed as a next-generation molecular diagnostics technology owing to its high sensitivity, specificity, and reliability11,12,13,14. These CRISPR/Cas diagnostics technologies rely on the collateral nuclease activity of Cas proteins to cleave single-stranded DNA (ssDNA) modified with a fluorescent reporter and a fluorescence quencher at each end of the oligonucleotides, as well as a fluorescence detection device to capture the released fluorescent reporter11,12. The nuclease activity of several Cas effectors activated by the CRISPR RNA (crRNA) target duplex can indiscriminately cleave the surrounding non-target ssDNA11. CRISPR-Cas12a (also called Cpf 1), a class 2 type V-A CRISPR/Cas system, demonstrates several advantages compared with Cas9, such as a lower mismatch tolerance and greater specificity13.&#160;The Cas12a/crRNA system has been applied for the sensitive and specific detection of the nucleic acids of human pathogens and phytopathogens14,15,16,17,18. Therefore, utilizing the Cas12a/crRNA system should enable the accurate and sensitive detection of the nucleic acid of CLas.
Cas12a alone is not theoretically sensitive enough to detect low levels of nucleic acids. Therefore, to improve its detection sensitivity, CRISPR-Cas12a detection is typically combined with an isothermal amplification step14,15. Recombinase polymerase amplification (RPA) enables sensitive and rapid isothermal DNA amplification in a temperature range from 37 &#176;C to 42 &#176;C19.
A detection platform called DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) that combines the DNase activity of Cas12a with RPA and a fluorescence readout has been recently devised12 and has been shown to detect nucleic acid with higher sensitivity20. Furthermore, the fluorescence signal emitted from the positive samples can be observed through a handheld fluorescence detection device in the field.
Since we amplified DNA with RPA, designed crRNA targeting the five-copy nrdB (ribonucleotide reductase &#946;- subunit) gene specific to CLas21, and employed the DNase activity of the Cas12a protein, we called this CLas detection method CLas-DETECTR. Compared with existing CLas detection methods, CLas-DETECTR is fast, accurate, sensitive, and deployable.

<table><tbody><tr><td>AxyPrep DNA Gel Extraction Kit</td><td>Corning</td><td>09319KE1</td><td>China</td></tr><tr><td>Bacterial Genomic DNA Extraction Kit</td><td>Solarbio</td><td>D1600</td><td>China</td></tr><tr><td>EnGen LbCas12a&amp;nbsp;</td><td>TOLOBIO</td><td>32104-01</td><td>China</td></tr><tr><td>Ex Taq Version 2.0 plus dye</td><td>TaKaRa</td><td>RR902A</td><td>China</td></tr><tr><td>Handheld fluorescent detection device&amp;nbsp;</td><td>LUYOR</td><td>3415RG</td><td>China</td></tr><tr><td>Hole puncher&amp;nbsp;</td><td>Deli</td><td>114</td><td>China</td></tr><tr><td>Magnesium acetate, MgOAc</td><td>TwistDx</td><td>TABAS03KIT</td><td>UK</td></tr><tr><td>NaOH</td><td>SCR</td><td>10019718</td><td>China</td></tr><tr><td>NEB buffer 3.1&amp;nbsp;</td><td>NEB</td><td>B7203</td><td>USA</td></tr><tr><td>PCR strip tubes</td><td>LABSELECT</td><td>PST-0208-FT-C</td><td>China</td></tr><tr><td>PEG 200&amp;nbsp;</td><td>Sigma</td><td>P3015</td><td>USA</td></tr><tr><td>PrimeSTAR Max DNA Polymeras</td><td>TaKaRa</td><td>R045A</td><td>China</td></tr><tr><td>Quick-Load Purple 1 kb Plus DNA Ladder&amp;nbsp;</td><td>New England Biolabs</td><td>N0550S</td><td>USA</td></tr><tr><td>TB Green Premix Ex Taq II (Tli RNaseH Plus)</td><td>TaKaRa</td><td>RR820B</td><td>China</td></tr><tr><td>TwistAmp Basic</td><td>TwistDx</td><td>TABAS03KIT</td><td>UK</td></tr></tbody></table>

field-deployable candidatus liberibacter asiaticus detection using recombinase polymerase amplification combined with crispr-cas12a

The early detection of Candidatus Liberibacter asiaticus (CLas) by citrus growers facilitates early intervention and prevents the spread of disease. A simple method for rapid and portable Huanglongbing (HLB) diagnosis is presented here that combines recombinase polymerase amplification and a fluorescent reporter utilizing the nuclease activity of the clustered regularly interspaced short palindromic repeats/CRISPR-associated 12a (CRISPR-Cas12a) system. The sensitivity of this technique is much higher than PCR. Furthermore, this method showed similar results to qPCR when leaf samples were used. Compared with conventional CLas detection methods, the detection method presented here can be completed in 90 min and works in an isothermal condition that does not require the use of PCR machines. In addition, the results can be visualized through a handheld fluorescent detection device in the field.

Here, we have described a portable platform, CLas-DETECTR, combing the RPA and CRISPR-Cas12a systems to diagnose HLB in the field. The schema of CLas-DETECTR is illustrated in Figure 1A.
When leaf samples from the HLB-infected and HLB-uninfected Newhall trees (Figure 1B), for which the presence of CLas was confirmed by PCR (Figure 1C), were subjected to the CLas-DETECTR test, a green fluorescence signal w...

Watch this Scientific Journal Video about Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a at JoVE.com

Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

In this work, a rapid, sensitive, and portable detection method for Candidatus Liberibacter asiaticus based on recombinase polymerase amplification combined with CRISPR-Cas12a was developed.

Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

The early detection of Candidatus Liberibacter asiaticus (CLas) by citrus growers facilitates early intervention and prevents the spread of disease. A ...

field-deployable-candidatus-liberibacter-asiaticus-detection-using

Research

JoVE Journal

Biology

2.5K Views.  Gannan Normal University. In this work, a rapid, sensitive, and portable detection method for Candidatus Liberibacter asiaticus based on recombinase polymerase amplification combined with CRISPR-Cas12a was developed.

Video: Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf mottling, vein yellowing, defoliation, root decay, and ultimately, the death of the citrus plant. When infected, the citrus plants have stunted growth and produce flowers out of season. These flowers rarely yield fruit, and those that do yield small, bitter, irregularly shaped citrus fruit that are not desirable. This disease is spread by the Asian citrus psyllid, Diaphorina citri, and by the grafting of infected citrus tissue. The pathogen has a long and variable incubation period within the citrus plant&#8212;sometimes years, before symptoms appear. Attempts to culture this pathogen in vitro have been unsuccessful, possibly due to the low and uneven concentration of the pathogen within infected citrus tissue, or because it is difficult to replicate the environmental conditions conducive to growth of the pathogen. It is very difficult to identify the disease before it has spread, due to its long incubation period and researchers&#39; inability to culture the pathogen. As a result, the disease only becomes apparent after suddenly destroying a citrus farmer&#39;s entire yield. Presented here is a method for the accurate and specific detection of the citrus greening pathogen, Candidatus liberibacter spp. using a genomic DNA extraction kit and PCR. This method is simple, efficient, cost effective, and adaptable for quantitative analysis. This method can be adapted for use on any citrus tissue; however, it is potentially limited by the amount of pathogen present in the tissue. Nevertheless, this method will allow citrus farmers to identify infected citrus plants earlier, and curb the spread of this destructive disease before it can further spread.

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter  spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus Greening is a particularly destructive disease affecting citrus crops globally. Presented here is a simple method using PCR and genomic DNA extraction of citrus leaf tissue for the accurate and precise identification of the citrus greening pathogen, Candidatus liberibacter spp.

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf ...

Environment

Molecular Evolution of the Tre Recombinase

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. 
We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of "molecular surgery" and molecular medicine.

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of molecular surgery and molecular medicine.

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within ...

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the avoidance of its potentially deleterious off-target effects. Several methods have been devised to reduce such effects. Here, we present an Escherichia coli-based directed evolution method called Sniper-screen to obtain a Cas9 variant with optimized specificity and retained on-target activity, called Sniper-Cas9. Using Sniper-screen, positive and negative selection can be performed simultaneously. The screen can also be repeated with other single-guide RNA (sgRNA) sequences to enrich for the true positive hits. By using the CMV-PltetO1 dual promoter to express Cas9 variants, the performance of the pooled library can be quickly checked in mammalian cells. Methods to increase the specificity of Sniper-Cas9 are also described. First, the use of truncated sgRNAs has previously been shown to increase Cas9 specificity. Unlike other engineered Cas9s, Sniper-Cas9 retains a wild-type (WT) level of on-target activity when combined with truncated sgRNAs. Second, the delivery of Sniper-Cas9 in a ribonucleoprotein (RNP) format instead of a plasmid format is possible without affecting its on-target activity.

Here, we present a protocol to optimize CRISPR-Cas9 to achieve a higher specificity without the loss of on-target activity. We use a directed evolution approach called Sniper-screen to find a mutant Cas9 with the desired characteristics. Sniper-Cas9 is compatible with truncated single-guide RNAs and delivery in a ribonucleoprotein format, well-known strategies for achieving higher specificities.

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the ...

Genetics

Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).

Here, we present a protocol describing a streamlined method for the efficient generation of plasmids expressing both the CRISPR enzyme and associated single guide RNA (sgRNAs). Co-transfection of mammalian cells with this sgRNA/CRISPR vector and a dual luciferase reporter vector that examines double-strand break repair allows evaluation of knockout efficiency.

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. ...

Bioengineering

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the CRISPR-Cas systems and their natural inhibitors-the anti-CRISPR proteins (Acrs)-provides a new tool to design and implement in vivo gene digital circuits. Here, we describe a protocol that follows the idea of the "Design-Build-Test-Learn" biological engineering cycle and makes use of dCas9/dCas12a together with their corresponding Acrs to establish small transcriptional networks, some of which behave like Boolean gates, in Saccharomyces cerevisiae. These results point out the properties of dCas9/dCas12a as transcription factors. In particular, to achieve maximal activation of gene expression, dSpCas9 needs to interact with an engineered scaffold RNA that collects multiple copies of the VP64 activation domain (AD). In contrast, dCas12a shall be fused, at the C terminus, with the strong VP64-p65-Rta (VPR) AD. Furthermore, the activity of both Cas proteins is not enhanced by increasing the amount of sgRNA/crRNA in the cell. This article also explains how to build Boolean gates based on the CRISPR-dCas-Acr interaction. The AcrIIA4 fused hormone-binding domain of the human estrogen receptor is the core of a NOT gate responsive to &#946;-estradiol, whereas AcrVAs synthesized by the inducible GAL1 promoter permits to mimic both YES and NOT gates with galactose as an input. In the latter circuits, AcrVA5, together with dLbCas12a, showed the best logic behavior.

CRISPR-Cas systems and anti-CRISPR proteins were integrated into the scheme of Boolean gates in Saccharomyces cerevisiae. The new small logic circuits showed good performance and deepened the understanding of both dCas9/dCas12a-based transcription factors and the properties of anti-CRISPR proteins.

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the ...

Optimized CRISPR-Based Molecular Diagnostics Protocol for Point-of-Care Use

Point-of-care CRISPR-based Diagnostics with Premixed and Freeze-dried Reagents

Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes that are suitable for use at point-of-care settings such as fast turnaround times for results, convenient simple readouts, and no requirement of complicated instruments. However, the reactions can be cumbersome to perform at the point of care due to their many components and manual handling steps. Herein, we provide a step-by-step, optimized protocol for the robust detection of disease pathogens and genetic markers with recombinase-based isothermal amplification and CRISPR-based reagents, which are premixed and then freeze-dried in easily stored and ready-to-use formats. Premixed, freeze-dried reagents can be rehydrated for immediate use and retain high amplification and detection efficiencies. We also provide a troubleshooting guide for commonly found problems upon preparing and using premixed, freeze-dried reagents for CRISPR-based diagnostics, to make the detection platform more accessible to the wider diagnostic/genetic testing communities.

Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases

Here, we present the step-by-step preparation of premixed, lyophilized recombinase-based isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based reactions, which can be used for the detection of nucleic acid biomarkers of infectious disease pathogens or other genetic markers of interest.

Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes ...

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of A. baumannii is crucial for prompt treatment, effective infection control, and curbing antibiotic resistance. However, there is no suitable method for rapid and easy on-site detection of A. baumannii. The DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) system offers a rapid, precise, and sensitive approach to A. baumannii detection by integrating the target-specific recognition capabilities of Cas12a with the isothermal amplification efficiency of Recombinase Polymerase Amplification (RPA). This protocol details the detection of A. baumannii using RPA combined with LbaCas12a endonuclease. The following steps are described in this article: extraction of DNA, selection of a specific DNA sequence, design of primer and CRISPR RNA (crRNA), construction of positive recombinant plasmid, setup of Cas12a-RPA assay, optimization of the RPA amplification system, visualization of the RPA-CRISPR/Cas12a assay using a fluorescence detection tool such as a real-time PCR instrument, and evaluation of sensitivity and specificity evaluation.

To facilitate the rapid and precise detection of Acinetobacter baumannii, we present a protocol that employs Recombinase Polymerase Amplification (RPA) in conjunction with LbaCas12a endonuclease for identifying A. baumannii infections.

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of ...

Immunology and Infection

DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning

We report a fast, easy-to-implement, highly sensitive, sequence-specific, and point-of-care (POC) DNA virus detection system, which combines recombinase polymerase amplification (RPA) and CRISPR/Cas12a system for trace detection of DNA viruses. Target DNA is amplified and recognized by RPA and CRISPR/Cas12a separately, which triggers the collateral cleavage activity of Cas12a that cleaves a fluorophore-quencher labeled DNA reporter and generalizes fluorescence. For POC detection, portable smartphone microscopy is built to take fluorescent images. Besides, deep learning models for binary classification of positive or negative samples, achieving high accuracy, are deployed within the system. Frog virus 3 (FV3, genera Ranavirus, family Iridoviridae) was tested as an example for this DNA virus POC detection system, and the limits of detection (LoD) can achieve 10 aM within 40 min. Without skilled operators and bulky instruments, the portable and miniature RPA-CRISPR/Cas12a-SPM with artificial intelligence (AI) assisted classification shows great potential for POC DNA virus detection and can help prevent the spread of such viruses.

We present a protocol that combines recombinase polymerase amplification with a CRISPR/Cas12a system for trace detection of DNA viruses and builds portable smartphone microscopy with an artificial intelligence-assisted classification for point-of-care DNA virus detection.

We report a fast, easy-to-implement, highly sensitive, sequence-specific, and point-of-care (POC) DNA virus detection system, which combines recombinase ...

Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

Summary

Explore More Videos

Chapters in this video

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Principles of Site-Specific Recombinase (SSR) Technology

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Molecular Evolution of the Tre Recombinase

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Point-of-care CRISPR-based Diagnostics with Premixed and Freeze-dried Reagents

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning