We want to thank all the students of the Synthetic Biology lab-SPST, TJU-for their general help, together with Zhi Li and Xiangyang Zhang for their assistance in FACS experiments.

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The authors declare no competing financial interest.

The protocol showed a possible complete workflow for synthetic gene digital circuits, following the &#34;Design-Build-Test-Learn&#34; (DBTL) biological engineering cycle and concerning both dry-lab and wet-lab experiments. Here, we focused on the CRISPR-Cas system, mainly dSpCas9, denAsCas12a, dLbCas12a, and the corresponding anti-CRISPR proteins, by designing and building in S. cerevisiae small transcriptional networks. Some of them mimicked Boolean gates, which are the basic components of digital circuits. All circuits described here permitted us to depict the properties and features of CRISPR-associated and anti-CRISPR proteins in S. cerevisiae. These results are essential to include these proteins in the scheme of gene digital circuits.
The DBTL concept provides a framework in synthetic biology, while many optimizations and improvements shall be made after testing a new artifact. For instance, in circuit 1, there was initially only one target site (one copy of lexOp) for dCas9/dCas12a-AD on the synthetic promoter upstream of yEGFP. After testing that circuit configuration, we found that it could achieve no more than a twofold activation15,16. Then, we assumed that by increasing the number of copies of lexOp, as in7, we could reach a higher transcriptional activation. Indeed, a higher fluorescence level was obtained by using three to six lexOp sites (Figure 1). Moreover, we further improved the performance of the circuits hosting dSpCas9 by engineering an scRNA, which is easier than fusing one or more ADs to a big protein like dSpCas9 (Figure 3). Additionally, by using a promoter of different strengths to produce the three AcrIIAs that we chose, we concluded that AcrIIA4 was the strongest inhibitor among them. Thus, we built a new NOT gate responsive to &#946;-estradiol by fusing the HBD(ER) to AcrIIA4 and exploiting the strong repression of AcrIIA4 on our best dSpCas9-based activator (Figure 4).
Similarly, we deeply characterized both the working of denAsCas12a and dLbCas12a in yeast and their interactions with three AcrVAs (Figure 5). For each dCas12a-AcrVA pair, we built a NOT (dCas12a was fused to an AD) and a YES (bare dCas12a) gate responsive to galactose. On the whole, dLbCas12a, together with AcrVA5, resulted in the best system to compute simple logic functions.
The method described here presented some critical steps. All protein DNA sequences were yeast codon-optimized to ensure higher expression in S. cerevisiae. To avoid unspecific targets of dSpCas9/denAsCas12a/dLbCas12a in the S. cerevisiae genome, we selected a bacterial operator such as lexOp. Moreover, strains containing the GAL1 promoter showed a significant growth delay that could limit the applicability of pGAL1 to synthetic gene circuits15.
Some modifications could also be brought to some steps in the overall method. In order to improve the efficiency of the digestion-ligation procedure, it is preferable to digest 10 &#181;g (overnight) of both the insert-containing plasmid and the acceptor vectors, rather than only 5 &#181;g in 1 h. In this way, a higher DNA concentration is reached after the elution step. The time for T4-ligation should be extended from 1 h (manufacturer&#39;s protocol) to 8 h. Finally, strains containing the dCas12a-VPR fusion protein should be diluted after a 24 h culture and grown for a further 12 h before running a FACS experiment. Under this condition, the variability among the fluorescence levels from different cells is no longer too high, and an acceptable standard deviation accompanies the mean value of the fluorescence intensity over a cell population.
In summary, this protocol explained how to simplify the design of gene digital circuits by making use of dCas proteins and, possibly, anti-CRISPR proteins. More importantly, we showed in detail how these families of proteins work in S. cerevisiae and which of them are the most promising for a future usage inside digital networks. An unsolved problem is the coupling of CRISPR-dCas/anti-CRISPR systems and chemicals, which represent the circuit inputs and cannot directly bind dCas proteins or anti-CRISPRs. Here, we bypassed the problem by using either the inducible GAL1 promoter or the HBD(ER) attached to AcrIIA4. However, a way to generalize the architecture of the circuit input layer is necessary to design synthetic gene digital circuits for different bioengineering areas such as metabolic engineering, biosynthesis, biosensing, biodiagnostics, and bioremediation.

In 2011, researchers proposed a computational method and developed a corresponding piece of software for the automatic design of digital synthetic gene circuits1. A user had to specify the number of inputs (three or four) and fill in the circuit truth table; this provided all the necessary information to derive the circuit structure using techniques from electronics. The truth table was translated into two Boolean formulae via the Karnaugh map method2. Each Boolean formula is made of clauses that describe logic operations (sum or multiplication) among (part of) the circuit inputs and their negations (the literals). Clauses, in their turn, are either summed up (OR) or multiplied (AND) to compute the circuit output. Every circuit can be realized according to any of its two corresponding formulae: one written in POS (product of sums) form and the other in SOP (sum of products) representation. The former consists of a multiplication of clauses (i.e., Boolean gates) that contain a logic sum of the literals. The latter, in contrast, is a sum of clauses where the literals are multiplied.
Electric circuits can be realized, on a breadboard, by physically wiring different gates together. The electric current permits the exchange of signals among gates, which leads to the computation of the output.
In biology, the situation is more complex. A Boolean gate can be realized as a transcription unit (TU; i.e., the sequence &#34;promoter-coding region-terminator&#34; inside eukaryotic cells), where transcription or translation (or both) are regulated. Thus, at least two kinds of molecules establish a biological wiring: the transcription factor proteins and the non-coding, antisense RNAs1.
A gene digital circuit is organized into two or three layers of gates, namely: 1) the input layer, which is made of YES (buffer) and NOT gates and converts the input chemicals into wiring molecules; 2) the internal layer, which consists of as many TUs as there are clauses in the corresponding Boolean formula. If the circuit is designed according to the SOP formula, every clause in the internal layer will produce the circuit output (e.g., fluorescence) in a so-called distributed output architecture. If the product of sum (POS) formula is used, then a 3) final layer is required, which will contain a single multiplicative gate collecting the wiring molecules from the internal layer.
Overall, in synthetic biology, many different schemes can be designed for the same circuit. They differ in the number and the kind of both TUs and wiring molecules. In order to choose the easiest solution to be implemented in yeast cells, each circuit design is associated with a complexity score S, defined as
<img alt="Equation 1" src="/files/ftp_upload/64539/64539eq01.jpg" style="margin: auto;" />
where A represents the number of activators, R represents the number of repressors, and a is the amount of antisense RNA molecules. If either activators or repressors are absent from the circuit, their contribution to S is zero. Therefore, it is more difficult to realize a circuit scheme in the lab (high S) when it requires a high number of orthogonal transcription factors. This means that new activators and repressors shall be engineered de novo in order to realize the complete wiring inside the digital circuits. In principle, novel DNA-binding proteins can be assembled by using Zinc Finger proteins3 and TAL effectors4 as templates. However, this option appears too arduous and time-consuming; therefore, one should rely mostly on small RNAs and translation regulation to finalize complex gene circuits.
Originally, this method was developed to fabricate digital circuits in bacteria. Indeed, in eukaryotic cells, instead of antisense RNAs, it is more suitable to talk of microRNAs (miRNAs) or small interfering RNAs (siRNAs)5. However, the RNAi pathway is not present in the yeast S. cerevisiae. Hence, one should opt for fully transcriptional networks. Suppose that a circuit needs five activators and five repressors; its complexity score would be S = 32. Circuit complexity can be reduced by replacing the 10 transcription factors with a single dCas96 (nuclease deficient Cas9) fused to an activation domain (AD). As shown in7, dCas9-AD works as a repressor in yeast when binding a promoter between the TATA box and the TSS (transcription start site) and as an activator when binding well upstream of the TATA box. Thus, one can replace 10 transcription factors with a single dCas9-AD fusion protein and 10 sgRNAs (single guide RNAs) for a total complexity score of S = 11. It is quick and easy to synthesize ten sgRNAs, whereas, as previously commented, the assembly of 10 proteins would demand much longer and more complicated work.
Alternatively, one might use two orthogonal dCas proteins (e.g., dCas9 and dCas12a): one to fuse to an AD, and the other bare or in combination with a repression domain. The complexity score would increase by only one unit (S = 12). Hence, CRISPR-dCas systems are the key to the construction of very intricate gene digital circuits in S. cerevisiae.
This paper deeply characterizes the efficiency of both dCas9- and dCas12a-based repressors and activators in yeast. Results show that they do not demand a high amount of sgRNA to optimize their activity, so episomal plasmids are preferentially avoided. Moreover, dCas9-based activators are far more effective when using a scaffold RNA (scRNA) that recruits copies of the VP64 AD. In contrast, dCas12a works well when fused to the strong VPR AD directly. Furthermore, a synthetic activated promoter demands a variable number of target sites, depending on the configuration of the activator (e.g., three when using dCas12a-VPR, six for dCas9-VP64, and only one with dCas9 and a scRNA). As a repressor, dCas12a appears more incisive when binding the coding region rather than the promoter.
As a drawback, however, CRISPR-dCas9/dCas12a do not interact with chemicals directly. Therefore, they might be of no use in the input layer. For this reason, alternative Boolean gate designs containing anti-CRISPR proteins (Acrs) have been investigated. Acrs act on (d)Cas proteins and inhibit their working8. Hence, they are a means to modulate the activity of CRISPR-(d)Cas systems. This paper thoroughly analyzes the interactions between type II Acrs and (d)Cas9, as well as type V Acrs and (d)Cas12a in S. cerevisiae. Since Acrs are much smaller than Cas proteins, a NOT gate responsive to the estrogen &#946;-estradiol was built by fusing the hormone-binding domain of the human estrogen receptor9-HBD(hER)-to AcrIIA4. Besides, a handful of YES and NOT gates that expressed dCas12a(-AD) constitutively and AcrVAs upon induction with galactose were realized. At present, these gates serve only as a proof of concept. However, they also represent the first step toward a deep rethinking of the algorithm to carry out the computational automatic design of synthetic gene digital circuits in yeast cells.

<table><tbody><tr><td>0.1 mL PCR 8-strip tubes</td><td>NEST</td><td>403112</td><td></td></tr><tr><td>0.2 mL PCR tubes</td><td>Axygen</td><td>PCR-02-C</td><td></td></tr><tr><td>1.5 mL Microtubes</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr><tr><td>15 mL Centrifuge tubes&amp;nbsp;</td><td>BIOFIL</td><td>CFT011150</td><td></td></tr><tr><td>2 mL Microtubes</td><td>Axygen</td><td>MCT-200-C&amp;nbsp;</td><td></td></tr><tr><td>50 mL Centrifuge tubes&amp;nbsp;</td><td>BIOFIL</td><td>CFT011500</td><td></td></tr><tr><td>Agarose-molecular biology grade</td><td>Invitrogen</td><td>75510-019</td><td></td></tr><tr><td>Ampicillin sodium salt</td><td>Solarbio</td><td>69-52-3</td><td></td></tr><tr><td>Applied biosystems veriti 96-well thermal cycler</td><td>Thermo Fisher Scientific</td><td>4375786</td><td></td></tr><tr><td>AxyPrep DNA gel extraction kit</td><td>Axygen</td><td>AP-GX-250</td><td></td></tr><tr><td>BD FACSuite CS&amp;amp;T research beads</td><td>BD</td><td>650621</td><td>Fluorescent beads</td></tr><tr><td>BD FACSVerse flow cytometer&amp;nbsp;</td><td>BD</td><td>-</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Centrifuge Sorvall ST 16R</td><td>Thermo Fisher Scientific</td><td>75004380</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; competent cells (Strain DH5&amp;alpha;)</td><td>Life Technologies</td><td>18263-012</td><td></td></tr><tr><td>ECL select Western Blotting detection reagent</td><td>GE Healthcare</td><td>RPN2235</td><td></td></tr><tr><td>Electrophoresis apparatus</td><td>Beijing JUNYI Electrophoresis Co., Ltd</td><td>JY300C</td><td></td></tr><tr><td>Flat 8-strip caps</td><td>NEST</td><td>406012</td><td></td></tr><tr><td>Gene synthesis company</td><td>Azenta Life Sciences</td><td></td><td>https://web.azenta.com/zh-cn/azenta-life-sciences</td></tr><tr><td>Goat anti-Mouse IgG (H+L) cross-adsorbed secondary antibody Alexa Fluor 568</td><td>Invitrogen</td><td>A-11004</td><td></td></tr><tr><td>HiFiScript cDNA synthesis kit</td><td>CWBIO</td><td>CW2569M</td><td>Kit used in step 6.2.2.1</td></tr><tr><td>Lysate solution (Zymolyase)</td><td>zymoresearch</td><td>E1004-A</td><td></td></tr><tr><td>Nikon Eclipse 80i fluorescence microscope</td><td>Nikon</td><td>-</td><td>Fluorescence microscope</td></tr><tr><td>Pipet tips&amp;mdash;10 &amp;mu;L</td><td>Axygen</td><td>T-300-R-S</td><td></td></tr><tr><td>Pipet tips&amp;mdash;1000 &amp;mu;L</td><td>Axygen</td><td>T-1000-B-R-S</td><td></td></tr><tr><td>Pipet tips&amp;mdash;200 &amp;mu;L</td><td>Axygen</td><td>T-200-Y-R-S</td><td></td></tr><tr><td>pRSII403</td><td>Addgene</td><td>35436</td><td></td></tr><tr><td>pRSII404</td><td>Addgene</td><td>35438</td><td></td></tr><tr><td>pRSII405</td><td>Addgene</td><td>35440</td><td></td></tr><tr><td>pRSII406</td><td>Addgene</td><td>35442</td><td></td></tr><tr><td>pRSII424</td><td>Addgene</td><td>35466</td><td></td></tr><tr><td>pTPGI_dSpCas9_VP64&amp;nbsp;</td><td>Addgene</td><td>49013</td><td></td></tr><tr><td>Q5 High-fidelity DNApolymerase</td><td>New England Biolabs</td><td>M0491</td><td></td></tr><tr><td>Restriction enzyme-Acc65I</td><td>New England Biolabs</td><td>R0599</td><td></td></tr><tr><td>Restriction enzyme-BamHI</td><td>New England Biolabs</td><td>R0136</td><td></td></tr><tr><td>Restriction enzyme-SacI-HF</td><td>New England Biolabs</td><td>R3156</td><td></td></tr><tr><td>Restriction enzyme-XhoI</td><td>New England Biolabs</td><td>R0146</td><td></td></tr><tr><td>Roche LightCycler 96&amp;nbsp;</td><td>Roche</td><td>-</td><td>Real-Time PCR Instrument</td></tr><tr><td>&lt;em&gt;S. cerevisiae&lt;/em&gt; CEN.PK2-1C</td><td>-</td><td>-</td><td>The parent strain. The genotype is: MATa; his3D1; leu2-3_112; ura3-52; trp1-289;&lt;br/&gt; MAL2-8c; SUC2</td></tr><tr><td>Stem-Loop Kit</td><td>SparkJade</td><td>AG0502</td><td>Kit used in step 6.2.1.3</td></tr><tr><td>T100 Thermal Cycler</td><td>BIO-RAD</td><td>186-1096</td><td></td></tr><tr><td>T4 DNA ligase</td><td>New England Biolabs</td><td>M0202</td><td></td></tr><tr><td>T5 Exonuclease</td><td>New England Biolabs</td><td>M0363</td><td></td></tr><tr><td>Taq DNA ligase</td><td>New England Biolabs</td><td>M0208</td><td></td></tr><tr><td>Taq DNA polymerase</td><td>New England Biolabs</td><td>M0495</td><td></td></tr><tr><td>TB Green Premix Ex Taq II (Tli RNaseH Plus)(2x) (SYBR Green I dye)</td><td>Takara</td><td>RR820Q</td><td></td></tr><tr><td>YeaStar RNA kit</td><td>Zymo Research</td><td>R1002</td><td></td></tr><tr><td>&amp;beta;-estradiol</td><td>Sigma-Aldrich</td><td>E8875</td><td></td></tr></tbody></table>

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.

sgRNA/crRNA expression by an RNA polymerase III-type promoter 
First, this work addressed the engineering of the transcriptional activation circuit (circuit 1) shown in Figure 1A. It contained three basic components: 1) the gene encoding for yEGFP (the reporter), which was preceded by a series of different synthetic promoters that provided target sites for dCas9/dCas12a-AD; 2) a yeast codon-optimized version of dCas9 or dCas12a fused to an activation domain (VP64 and VPR, respectively) and containing either one or two nuclear localization sequences (NLSs). Both dCas proteins were produced by a strong constitutive promoter-pGPD; and 3) an sgRNA/crRNA sequence that guided dCas9/dCas12a-AD to the target sites. The activation efficiency of dCas9/dCas12a-based activators was visualized and reflected by the fluorescence intensity of the reporter (measured via FACS experiments).
One dCas9 protein (dSpCas9) and two dCas12a proteins (denAsCas12a and dLbCas12a) were tested. A 3.36-fold activation was achieved with dSpCas9 extended, at its C terminus, with the VP64 AD and binding a synthetic promoter-upstream of yEGFP-containing six copies of the lexOp target site. The sgRNA was placed into an integrative shuttle vector and transcribed by the RNA polymerase III-dependent SNR52 promoter (SNR52i configuration, see Figure 1B). In the case of dCas12a, denAsCas12a-VPR returned the highest activation (4.45-fold) from a synthetic promoter with three operators when the crRNA was expressed via the SNR52i configuration (Figure 1C). Under the same conditions, dLbCas12a-VPR achieved its best fluorescence enhancement (3.21-fold) (Figure 1D). It should be noted that the comparison term, in every experiment, was a circuit whose sgRNA/crRNA could not bind the lex operators.
Multicopy plasmids are not necessary 
The SNR52i sgRNA expression cassette was replaced with an RGR structure expressed by a moderately strong promoter-pADH1. However, in both dCas9 and dCas12a cases, activation in the presence of an sgRNA/crRNA generated by the self-cleavage of RGR appeared comparable to or even lower than that achieved with an sgRNA/crRNA produced via SNR52i, despite the fact that pSNR52 was considered as a weak promoter (see Figure 1C,D for the first results obtained with dCas12a).
To further explore the connection between sgRNA/crRNA amount and activation efficiency, the two sgRNA/crRNA expression systems were inserted into an episomal plasmid, which can be taken up by the cell in 10-40 copies and generate a greater quantity of sgRNA/crRNA. As shown in Figure 2A, the activation by crRNA located on an integrative plasmid (either SNR52i or RGRi) was 1.4- to 2.4-fold higher than that achieved when the same crRNA was expressed by an episomal plasmid (either SNR52m or RGRm). The trend was confirmed by the sgRNA. In this case, the integrative plasmid guaranteed a 1.1- to 1.5-fold higher activation (Figure 2B). To exclude that the results were caused by a loss of the episomal plasmids, RT-qPCR was carried out to quantify the sgRNA/crRNA relative abundance in vivo. The results, in Figure 2C,D, verified that the episomal vector produced a much higher level of sgRNA/crRNA than the integrative vector, no matter the expression system (RGR or SNR52). These results showed that the SNR52 system could work even better than the RGR system, and a higher amount of sgRNA/crRNA in the cell did not guarantee a higher activation by the CRISPR-Cas system. Therefore, episomal plasmids should not be employed in the construction of gene digital circuits where dCas9/dCas12a-AD are used.
Scaffold RNA engineering 
An scRNA was engineered by extending the sequence of an sgRNA with MS2 hairpin structures that permitted to recruit VP64 AD when fused to the MS2 coat protein (MCP, see Figure 3A). In this way, no engineering of, nor modifications on, dCas9 were necessary. Two MS2 variants were tried: wt and f6. The scRNA containing the single MS2 hairpin-1&#215;MS2(wt) and 1&#215;MS2(f6)-gave a 5.27-fold and a 4.34-fold activation, respectively. However, the scRNA with a combination of the two hairpins-2&#215;MS2(wt+f6)-returned the overall highest activation in this study (7.54-fold, see Figure 3B). These results demonstrated that engineering an scRNA was far more effective than fusing any activation domains to dSpCas9 directly.
Boolean gate based on Acr proteins 
To further control and tune transcriptional activation by CRISPR-Cas systems, circuit 1 was modified with the insertion of a fourth TU for the expression of anti-CRISPR proteins (see Figure 4A for AcrIIAs and Figure 5A for AcrVAs). After showing that Acrs were effective, in S. cerevisiae, in contrasting the activation due to dCas9/dCas12a-AD, a new circuit was built (see Figure 5D) to test AcrVA action on dCas12a-based repressors (a previous work33 had already shown that AcrIIAs could inhibit dCas9-based gene down-regulation). The new, Acr-containing small networks worked as simple Boolean gates (YES and NOT), which might lead to a restyling of the input layer of more complex synthetic gene digital circuits.
AcrIIA4 is a strong inhibitor of dSpCas9 
Four different promoters with diverse strengths were used to drive the expression of three kinds of AcrIIs-AcrIIA2, AcrIIA4, and AcrIIA5. The results showed that the three AcrIIs worked in a dose-dependent manner in S. cerevisiae. When expressed by a strong promoter-pGPD-they reduced the fluorescence level reached by dSpCas9:scRNA_2&#215;MS2(wt+f6)-MCP-VP64 to 0.21, 0.11, and 0.13 of its value, respectively (Figure 4B). Since AcrIIA4 was the only one that caused high inhibition of fluorescence expression even when produced by a weak, synthetic promoter-genCYC1t_pCYC1noTATA-we could infer that AcrIIA4 was the strongest inhibitor among the three AcrIIs. Next, the HBD(ER) was fused to the C terminus of AcrIIA4 to build a &#946;-estradiol sensing device (see Figure 4C). In the presence of the estrogen &#946;-estradiol, AcrIIA4-HBD(ER) could translocate into the nucleus and then neutralize the function of the dSpCas9-based activator. The titration curve in Figure 4D shows that the circuit behaves like a NOT gate with an ON/OFF ratio approaching 2.3.
AcrVAs are repressors of dCas12a proteins 
A NOT gate was designed and built by inserting the pGAL1-driving AcrVA expression cassette into circuit 1. In this way, AcrVA synthesis, and the consequent repression of dCas12a-AD, could be induced by galactose (Figure 5A).&#160;As shown in Figure 5B,C, AcrVA1 hindered both denAsCas12a and dLbCas12a as activators by reducing fluorescence expression from 19% to 71%, depending on the circuit scheme. AcrVA4 and AcrVA5 cannot exert any action on denAsCas12a34. However, they performed a strong inhibition of dLbCas12a-based activators by reducing fluorescence expression up to 84% (AcrVA5) and 82% (AcrVA4). On the whole, AcrVA5 turned out to be the most reliable, among these three AcrVAs, in inhibiting dLbCas12a-based activators because it guaranteed, in different circuits, higher than 70% repression.
AcrVA1 action is concentration-dependent 
The relation between AcrVAs&#39; in vivo concentration and their inhibitory effects on both activators and repressors based on denAs/dLbCas12a was also studied. To this aim, each AcrVA was expressed under different promoters: the strong pGAL1, the medium-strength pTEF1, and the weak genCYC1t_pCYC1noTATA. As illustrated in Figure 5E, AcrVA1 displayed big fluctuations in its performance depending on the promoter that led its synthesis. AcrVA1 worked reasonably well only when produced by pGAL1. Under pTEF1 and genCYC1t_pCYC1noTATA, AcrVA1 showed some repression on the bare dLbCas12a only. AcrVA4 and AcrVA5, in contrast, appeared to be less sensitive to their concentration, especially when interacting with the bare dLbCas12a.
This data showed that AcrVA4 and AcrVA5 generally performed better than AcrVA1 in inhibiting dLbCas12a-based transcription factors in S. cerevisiae. It should be noted that AcrVA5 has a peculiar working mechanism, since it acts as an enzyme that modifies LbCas12a permanently. However, as mentioned above, AcrVA5 (together with AcrVA4) cannot interact with denAsCas12a.
When AcrVAs were expressed under pGAL1, circuits become either YES gates (dCas12a was fused to an AD) or NOT gates (bare dCas12a). The former looked all highly performant, while the latter appeared to work better in the presence of AcrVA4 or AcrVA5.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64539/64539fig01.jpg" /> 
Figure 1: Transcriptional activation mediated by dCas9/dCas12a-AD. (A) Circuit 1 diagram. The synthetic promoters of yEGFP contained six (6x) and three (3x) copies of target sites for dCas9 or dCas12a, respectively. After that, dCas9/dCas12a-AD combines with sgRNA/crRNA; the synthetic promoter is targeted and activated due to the presence of the activation domains. (B) The best activation efficiency of dSpCas9-VP64 was achieved when there were six lexOp target sites on the synthetic promoter, and the sgRNA was transcribed by SNR52i. (C,D) The highest activation efficiency of dCas12a-VPR was obtained when three copies of lexOp were inserted into the synthetic promoter, and the crRNA was generated by SNR52i. SNR52i means that the sgRNA/crRNA was produced by pSNR52, and the expression cassette was placed inside an integrative shuttle vector. RGRi means an integrative plasmid hosting the RGR cassette to express sgRNA/crRNA was used. The negative control, &#34;-&#34;, represents a sgRNA/crRNA containing a scrambled spacer sequence that does not match with the lexOp site nor with any sequence along the yeast genome. &#34;bA&#34; indicates that the sgRNA/crRNA binds the antisense strand of target DNA, while &#34;bS&#34; stands for binding the sense strand. Each fluorescence level represents the mean value from at least three independent experiments (i.e., carried out on different days). Error bars are the standard deviation of the mean. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64539/64539fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64539/64539fig02.jpg" /> 
Figure 2: The comparison of integrative and episomal plasmid-producing sgRNA/crRNA. (A) Activation efficiency of dLbCas12a-VPR:crRNA when targeting a single site on the promoter upstream of yEGFP. (B) dSpCas9-VP64:sgRNA activated n&#215; synthetic promoter (&#34;n&#34; stands for the number of lexOp target sites). (C,D) Normalized sgRNA/crRNA expression level15,16. &#34;i&#34; means that the sgRNA/crRNA expression cassette was placed into an integrative shuttle vector, while &#34;m&#34; stands for multicopy (i.e., episomal) plasmid. &#34;bA&#34;/&#34;bS&#34; indicates that the sgRNA/crRNA binds the antisense/sense strand of the DNA. &#34;ctrl&#34; is a negative control, where a scrambled sgRNA/crRNA was expressed. Each fluorescence level represents the mean value from at least three independent experiments (i.e., carried out on different days). Error bars are the standard deviation of the mean. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64539/64539fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64539/64539fig03.jpg" /> 
Figure 3: The activation efficiency of the bare dSpCas9 in complex with an scRNA. (A) The schematic diagram of the interactions among scRNA, MCP-VP64, and the bare dSpCas916. The cap-like structure in purple represents MCP (MS2 coat protein). One MS2 hairpin (the purple structure in the scRNA) can recruit and bind two copies of MCP. Thus, an scRNA enables not only DNA-binding by dSpCas9 but also activation of gene expression through the recruitment of MCP-VP64. (B) The activation efficiency of dSpCas9:scRNA-MCP-VP64. Three kinds of scRNA were tested: one carrying the wild-type MS2 hairpin-1&#215;MS2(wt), another engineered with the f6 MCP aptamer-1&#215;MS2(f6), and the last one containing both hairpins-2&#215;MS2(wt+f6), which turned out to be the most performant. Each fluorescence level represents the mean value from at least three independent experiments (i.e., carried out on different days). Error bars are the standard deviation of the mean. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64539/64539fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64539/64539fig04.jpg" /> 
Figure 4: AcrIIA-related circuits and results. (A) The AcrIIA expression cassette was inserted into circuit 1. This additional TU includes pGPD leading the expression of AcrIIAs to counteract dSpCas9:scRNA_2&#215;MS2(wt+f6)-MCP-VP64. (B) The inhibition efficiency of AcrIIAs on the best dSpCas9-based activator in Figure 3B. The black dashed line represents the fluorescence in the presence of the dSpCas9-based activator. The figures above each column show the inhibition efficiency calculated as the OFF/ON ratio (i.e., the fluorescence level in the presence of the AcrIIA divided by the fluorescence in the absence of any AcrIIA). In the legend, the strength of the four constitutive promoters gradually increases from up to down. (C) Diagram of the &#946;-estradiol sensing device (NOT gate) expressing AcrIIA4-HBD(hER). (D) Titration curve of the circuit in (C)16. The green curve refers to the change in fluorescence in the functional circuit. The black curve was derived from the strain without the expression of AcrIIA4-HBD(hER)-the negative control. The dashed line in gray marked the fluorescence plateau at the equilibrium. It was calculated as the mean of the fluorescence values at concentrations of &#946;-estradiol not lower than 125 nM. Each fluorescence level represents the mean value from at least three independent experiments (i.e., carried out on different days). Error bars are the standard deviation of the mean. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64539/64539fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64539/64539fig05.jpg" /> 
Figure 5: AcrVA-related circuits and results. (A) The AcrVA expression cassette was inserted into circuit 1. The new TU contains the inducible promoter pGAL1 upstream of the AcrVA genes that neutralize the working of dCas12a-AD. The new circuit is a NOT gate regulated by galactose. (B,C) Results from the galactose-responsive NOT gate in (A). Here, pGAL1 drives the synthesis of AcrVAs, which then interact with dCas12a-AD15. The relative fluorescence corresponds to the OFF/ON ratio. (D) Galactose-responsive YES gate. It employs AcrVAs under the control of pGAL1 and the bare dCas12as that represses the synthesis of yEGFP. (E) Comparison of the inhibition efficiencies of AcrVAs expressed by promoters of different strength15. The groups &#34;AcrV effects on repression&#34; and &#34;bare dLb&#34; refer to the circuit in (D). The groups &#34;AcrV effects on activation&#34; and &#34;dLb-VPR&#34; are the results of the NOT gate in (A). Each fluorescence level represents the mean value from at least three independent experiments (i.e., carried out on different days). Error bars are the standard deviation of the mean. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64539/64539fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Table 1: A list of all DNA sequences used in this study. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64539/Suplementary Table 1.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Table 2: A list of primers used in this study. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64539/Suplementary Table 2.xlsx" target="_blank">Please click here to download this File.</a>
Supplemental Coding Files: The R studio script to analyze the FCS files. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64539/BDverse_analysis.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins at JoVE.com

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

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 ...

gene-digital-circuits-based-on-crispr-cas-systems-anti-crispr

Nanyang Technological University

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Unit 1

DC Circuit Analysis

SCIENTISTS IN ACTION

1.7K Views.  Tianjin University. Ons protocol legt uit hoe digitale gistgencircuits kunnen worden ontworpen, gebouwd en geanalyseerd die gebruikmaken van type twee en type vijf CRISPR dCas-systemen en de bijbehorende anti-CRISPR-eiwitten. Het blijft assembleren omdat het nul standaardprocedures verzamelt om synthetische transcriptionele netwerken in services samen te stellen en te testen. Bereid om te beginnen een reactiemengsel voor met 20 tot 40 nanogram DNA-sjabloon, één microliter voorwaartse primer, één microliter o...

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

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

Genetics

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic engineering became applicable to a large range of systems. Moreover, many transcriptional and epigenomic engineering approaches are now generally feasible for the first time. One reason for the broad applicability of CRISPR lies in its bipartite nature. Small gRNAs determine the genomic targets of the complex, variants of the protein Cas9, and the local molecular consequences. However, many CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into individual cells. Here, we present a customizable protocol for string assembly gRNA cloning (STAgR), a method that allows the simple, fast and efficient generation of multiplexed gRNA expression vectors in a single cloning step. STAgR is cost-effective, since (in this protocol) the individual targeting sequences are introduced by short overhang primers while the long DNA templates of the gRNA expression cassettes can be re-used multiple times. Moreover, STAgR allows single step incorporation of a large number of gRNAs, as well as combinations of different gRNA variants, vectors and promoters.

A Customizable Protocol for String Assembly gRNA Cloning STAgR

Here, we present string assembly gRNA cloning (STAgR), a method to easily multiplex gRNA vectors for CRISPR/Cas9 approaches. STAgR makes gRNA multiplexing simple, efficient and customizable.

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (CRISPR/Cas9) technology has become a prevalent laboratory tool to introduce accurate and targeted modifications in the genome. Its enormous popularity and rapid spread are attributed to its easy use and accuracy compared to its predecessors. Yet, the constitutive activation of the system has limited applications. In this paper, we describe a new method that allows temporal control of CRISPR/Cas9 activity based on conditional stabilization of the Cas9 protein. Fusing an engineered mutant of the rapamycin-binding protein FKBP12 to Cas9 (DD-Cas9) enables the rapid degradation of Cas9 that in turn can be stabilized by the presence of an FKBP12 synthetic ligand (Shield-1). Unlike other inducible methods, this system can be adapted easily to generate bi-cistronic systems to co-express DD-Cas9 with another gene of interest, without conditional regulation of the second gene. This method enables the generation of traceable systems as well as the parallel, independent manipulation of alleles targeted by Cas9 nuclease. The platform of this method can be used for the systematic identification and characterization of essential genes and the interrogation of the functional interactions of genes in in vitro and in vivo settings.

Here, we describe a genome-editing tool based on the temporal and conditional stabilization of clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (Cas9) under the small molecule, Shield-1. The method can be used for cultured cells and animal models.

The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (CRISPR/Cas9) technology has become a prevalent laboratory ...

Detecting Off-Target CRISPR-Cas9 Cleavage Events with CIRCLE-Seq

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of unintended cleavage sites of CRISPR-Cas9 through targeted sequencing of CRISPR-Cas9 cleaved DNA. The protocol involves circularizing genomic DNA (gDNA), which is subsequently treated with the Cas9 protein and a guide RNA (gRNA) of interest. Following treatment, the cleaved DNA is purified and prepared as a library for Illumina sequencing. The sequencing process generates paired-end reads, offering comprehensive data on each cleavage site. CIRCLE-seq provides several advantages over other in vitro methods, including minimal sequencing depth requirements, low background, and high enrichment for Cas9-cleaved gDNA. These advantages enhance sensitivity in identifying both intended and unintended cleavage events. This study provides a comprehensive, step-by-step procedure for examining the off-target activity of CRISPR-Cas9 using CIRCLE-seq. As an example, this protocol is validated by mapping genome-wide unintended cleavage sites of CRISPR-Cas9 during the modification of the AAVS1 locus. The entire CIRCLE-seq process can be completed in two weeks, allowing sufficient time for cell growth, DNA purification, library preparation, and Illumina sequencing. The input of sequencing data into the CIRCLE-seq pipeline facilitates streamlined interpretation and analysis of cleavage sites.

A significant barrier to technologies like CRISPR is the off-target events that can disrupt vital genes. 'Circularization for In Vitro Reporting of Cleavage Effects by Sequencing' (CIRCLE-seq) is a technique designed to identify unintended cleavage sites. This method maps the genome-wide activity of CRISPR-Cas9 with high sensitivity and without bias.

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of ...

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

Neuroscience

Centromere-Associated Protein-E Gene Editing Using the CRISPR/Cas9 System

Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing in a variety of organisms. Centromere-associated protein-E (CENP-E) is a plus-end-directed kinesin required for kinetochore-microtubule capture, chromosome alignment, and spindle assembly checkpoint. Although cellular functions of the CENP-E proteins have been well studied, it has been difficult to study the direct functions of CENP-E proteins using traditional protocols because CENP-E ablation usually leads to spindle assembly checkpoint activation, cell cycle arrest, and cell death. In this study, we have completely knocked out the CENP-E gene in human HeLa cells and successfully generated the CENP-E-/- HeLa cells using the CRISPR/Cas9 system.
Three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, which effectively improve the screening efficiency and experimental success rate of the CENP-E knockout cells. Importantly, CENP-E deletion results in chromosome misalignment, the abnormal location of the BUB1 mitotic checkpoint serine/threonine kinase B (BubR1) proteins, and mitotic defects. Furthermore, we have utilized the CENP-E knockout HeLa cell model to develop an identification method for CENP-E-specific inhibitors.
In this study, a useful approach to validate the specificity and toxicity of CENP-E inhibitors has been established. Moreover, this paper presents the protocols of CENP-E gene editing using the CRISPR/Cas9 system, which could be a powerful tool to investigate the mechanisms of CENP-E in cell division. Moreover, the CENP-E&#160;knockout cell line would contribute to the discovery and validation of CENP-E inhibitors, which have important implications for antitumor drug development, studies of cell division mechanisms in cell biology, and clinical applications.

Author Spotlight: Establishing CENP-E Knockout HeLa Cells &#8211; A Novel Approach to Study Kinesin-7 CENP-E Biology and its Inhibitors

This article reports the construction of centromere-associated protein-E (CENP-E) knockout cells using the CRISPR/Cas9 system and three phenotype-based screening strategies. We have utilized the CENP-E&#160;knockout cell line to establish a novel approach to validate the specificity and toxicity of the CENP-E inhibitors, which is useful for drug development and biological research.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing ...

Biology

Genome Editing

A well-established technique for modifying specific sequences in the genome is gene targeting by homologous recombination, but this method can be laborious and only works in certain organisms. Recent advances have led to the development of &#8220;genome editing&#8221;, which works by inducing double-strand breaks in DNA using engineered nuclease enzymes guided to target genomic sites by either proteins or RNAs that recognize specific sequences. When a cell attempts to repair this damage, mutations can be introduced into the targeted DNA region. In this video, JoVE explains the principles behind genome editing, emphasizing how this technique relates to DNA repair mechanisms. Then, three major genome editing methods&#8212;zinc finger nucleases, TALENs, and the CRISPR-Cas9 system&#8212;are reviewed, followed by a protocol for using CRISPR to create targeted genetic changes in mammalian cells. Finally, we discuss some current research that applies genome editing to alter the genetic material in model organisms or cultured cells.

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

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