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CRISPRecise Kit
(Kit # 1000000235 )

Depositing Lab:   Ervin Welker

CRISPRecise is a set of increased-fidelity (also known as high-fidelity) SpCas9 variants that can be used to efficiently edit any SpCas9 target with practically no detectable off-target effects. The CRISPRecise collection comprises 17 variants, each offering incrementally higher levels of fidelity and correspondingly reduced overall activity. This increased-fidelity nuclease (IFN) set is designed to provide an optimal, target-matched IFN for every SpCas9 target. To ensure maximum specificity during editing, it is important to identify the most appropriate IFN for each target (i.e., the one with the highest fidelity and lowest activity that can still effectively cleave the target). Thanks to the cleavage rule, there is no need to assess the specificity of all 17 IFNs by genome-wide off-target detection methods. Since each target appears to have a unique sensitivity to cleavage, which determines the minimal amount of IFN activity required for efficient cleavage, the target-matched IFN can be found by a systematic search rather than a random one. This variant typically allows editing without detectable off-target effects and can be easily identified from the pool of 17 variants by assessing on-target activities.

This kit will be sent as bacterial glycerol stocks in 96-well plate format.

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$ 400 USD + shipping

Available to academics and nonprofits only.

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Original Publication

A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets. Kulcsar PI, Talas A, Ligeti Z, Toth E, Rakvacs Z, Bartos Z, Krausz SL, Welker A, Vegi VL, Huszar K, Welker E. Nat Commun. 2023 Sep 16;14(1):5746. doi: 10.1038/s41467-023-41393-5. PubMed (Link opens in a new window) Article (Link opens in a new window).

Description

The CRISPRecise collection contains 17 increased-fidelity SpCas9 variants, ranging from the lowest fidelity variant, the Blackjack-SpCas9, to the highest fidelity variant, the B-HeFSpCas9, providing an optimal target-matched variant (i.e., one that provides efficient editing with no detectable off-target effects) for virtually any SpCas9 target regardless of its cleavability.

To identify the target-matched variant for a given target, we have developed a two-step algorithm that eliminates the need to test all variants. The first step is to measure the on-target activity of the wildtype SpCas9 and three IFNs (e-plus, B-HF1, and B-HypaR) that divide the cleavage range of the targets into four proportional sections, to identify the variant with the highest fidelity that still maintains efficiency. For those who are not attempting to achieve maximum specificity, the results of this first screen may provide a satisfactory high specificity with few residual off-targets. In the second step, additional IFNs situated between the last efficiently working variant and the first non-working one (identified in the first step) are tested for on-target activity to identify the target-matched variant (Figure 1). For the vast majority of targets, this can be achieved without genome-wide off-target assessment thanks to the cleavage rule (Figure 2). Target sequence contribution (manifested in the cleavability of the target by an IFN), the effects of fidelity-increasing mutations in the IFNs, and mismatches are the three main factors that collectively determine whether an IFN will cleave a target or any of its off-targets.

On the one hand, the increased-fidelity variants can be ranked according to the effects of their fidelity-increasing mutations (i.e., their fidelity) and this order also reflects their overall on-target activity rank. The lower overall on-target activity is due to greater target selectivity (i.e., the variant cleaves fewer targets than the wildtype SpCas9) but often cleaves them with wildtype-like efficiency, resulting in a lower average/overall editing rate. On the other hand, each target can be ranked on their sensitivity to cleavage, not only determining which variants can cleave a given target, but strongly influencing the cleavability of potential off-target sequences for the corresponding gRNA. These two effects determine the target-matched IFN for a given target, where the inhibitory effect of the fidelity-increasing mutations is only slightly smaller than the activating effect of the target sequence, so that it can still effectively cleave the on-target sequence, but when the effect of a mismatch is added, the combined inhibitory effect exceeds the contribution of the target sequence, and therefore it does not cleave any off-target. A simplified illustration of the cleavage rule is presented in Figure 2.

Other factors may also influence on-target and off-target cleavage, however they typically exert minor effects, causing some deviation from the cleavage rule in a small percentage of cases.

The cleavage rule is apparent when using the variants in ribonucleoprotein (RNP) form as well, although the optimal variant could be slightly shifted compared to the plasmid experiments.

Schematic showing the two step approach for identifying the target-matched SpCas9 nuclease. The top panel, labelled, “Correlation between cleavage activity and specificity on a hypothetical target example” shows SpCas9 variants from left to right with increasing fidelity: WT (100-75% on-target efficiency with off-target detection by GUIDE-seq), Blackjack (75-50% with off-target), B-Sniper (100-75% with off-target), B-HiFi (75-50% with off-target), e (75-50% with NO off-target detection by GUIDE-seq), e-plus (100-75% with NO off-target), HF1-plus (75-50% with NO off-target), HF1, B-e (both 50-25%), Hypa, B-HF1, B-Hypa, HypaR, B-HypaR, evo, B-evo, HeF, and B-HeF (all 25-0%). An arrow pointing to the bottom panel reads, “Method for finding the optimal, target-matched variant on the same example without all variants.” The 1st on-target screen is a Rough screening by NGS and WT (100-75%), e-plus (100-75%), B-HF1 (25-0%), and B-HypaR (25-0%) were screened. The 2nd on-target screen is a Fine tuning step by NGS and focused on the five variants between the working e-plus (included in the 2nd screen) and the non-working B-HF1, that are HF1-plus (75-50%), HF1 (50-25%), B-e (50-25%) and Hypa (25-0%). The Off-target detection by GUIDE-seq found that e-plus and HF1-plus had NO off-targets.
  • Figure 1. The optimal, target-matched SpCas9 nuclease, which shows efficient on-target editing and no off-target effects is identified for each target using a two-step approach.

    The first panel shows the on- and off-target activity of a set of IFNs with increasing fidelity on a target. The second panel shows the screening method that identifies the optimal variant for the target without having to test all the variants. In the first step, a rough on-target screen is performed, where the WT and three specifically selected IFNs that divide the target ranking range into four approximately proportional sections, are tested. The second step is a fine-tuning on-target screen involving the not yet assessed variants with higher fidelity than the highest ranking active (green) variant from the first screen. The fine-tuning on-target screen identifies the target-matched variants (active variants with the highest fidelity). If necessary, two sufficiently active (here their normalized activity is above 50%, but this may depend on the application under consideration) target-matched variants can be screened for the absence of genome-wide off-targets. The figure is from the Kulcsár, et. al. Nature Communications article, reprinted under the terms of the CC-BY-NC (Link opens in a new window).

The left panel shows a four-quadrant plot for possible combinations of increased fidelity nucleases (IFNs) harboring mutations with increasing activity-diminishing effects and target sequences with increasing activating effect. The two top boxes have a high activating effect of the target sequence that ensure a WT-like IFN cleavage with both the low effect (top left box) and the higher effect (top right box) IFN mutations. The bottom two boxes have a medium to low effect of the target sequence that ensure a WT-like IFN cleavage only with low effect IFN mutations (bottom left box) but no cleavage with high effect IFN mutations (bottom right box). The right panel shows another four-quadrant plot that is exactly the same except the top left and right and bottom left boxes also have low effect of the mismatches and the bottom right box is blank and reads “Off-target not tested due to no on-target cleavage.” The top left box has high off-target cleavage and the top right and bottom left boxes have increasing specificity and no off-target cleavage.
  • Figure 2. Simplified explanatory figure of the 'cleavage rule'.

    The left panel shows the on-target activity of two IFNs from different fidelity ranks on two targets from different cleavability ranks. When the activating effect of the target sequence contribution is larger than the inhibitory effect of the fidelity-increasing mutations, the SpCas9 variant cleaves the target (blue background), but when it is smaller, the variant does not cleave the target (red background). The right panel shows the effect of a mismatch on the activity of the IFNs from the first panel on the same targets. When the activating effect of the target sequence contribution is larger than the combined inhibitory effect of the fidelity-increasing mutations and the mismatches, the SpCas9 variant cleaves the off-target sequence (yellow background), but when it is smaller, the variant does not cleave (burgundy background). The effect of the same mismatch can vary in different sequential contexts, but for simplicity, here we apply the same effect on all the example cases. The figure is from the Kulcsár, et. al. Nature Communications article, reprinted under the terms of the CC-BY-NC (Link opens in a new window).

Protocol

Protocol for a typical experiment to test the on-target activities of IFN variants:

In cases with different cell lines (e.g., N2a, HEK293), plate cells one day prior to transfection in 48-well plates at a density of approximately 2.5–3 × 104 cells/well. Co-transfect cells with two types of plasmids: SpCas9 variant expression plasmid (137 ng) and sgRNA and mCherry coding plasmid (97 ng) using 1 µL TurboFect reagent according to the manufacturer’s protocol. For negative control experiments, co-transfect either deadSpCas9 plasmid with a targeting sgRNA plasmid, or active SpCas9 variant with a non-targeting sgRNA plasmid. Calculate transfection efficacy via mCherry expressing cells. Analyze transfected cells ~96 h post transfection by flow cytometry, and purify genomic DNA for NGS analysis.

How to Cite this Kit

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.

For your Materials and Methods section:

“The CRISPRecise Kit was a gift from Ervin Welker (Addgene kit #1000000235).”

For your Reference section:

A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets. Kulcsar PI, Talas A, Ligeti Z, Toth E, Rakvacs Z, Bartos Z, Krausz SL, Welker A, Vegi VL, Huszar K, Welker E. Nat Commun. 2023 Sep 16;14(1):5746. doi: 10.1038/s41467-023-41393-5. PubMed (Link opens in a new window) Article (Link opens in a new window).

CRISPRecise Kit - #1000000235

Resistance Color Key

Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.

Inventory

Searchable and sortable table of all plasmids in kit. The Well column lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.

Kit Plate Map

96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.

Resistance Color Key

Ampicillin
Kanamycin

Inventory

Well Plasmid Resistance
A / 1 pX330-Flag-WT_SpCas9 (without sgRNA; with silent mutations)
Ampicillin
A / 2 pX330-Flag-eSpCas9 (without sgRNA; with silent mutations)
Ampicillin
A / 3 pX330-Flag-SpCas9-HF1 (without sgRNA; with silent mutations)
Ampicillin
A / 4 pX330-Flag-HypaSpCas9 (without sgRNA; with silent mutations)
Ampicillin
A / 5 HypaR-SpCas9
Ampicillin
A / 6 pX330-Flag-evoSpCas9 (without sgRNA; with silent mutations)
Ampicillin
A / 7 pX330-Flag-HeFSpCas9 (without sgRNA; with silent mutations)
Ampicillin
A / 8 B-SpCas9
Ampicillin
A / 9 B-eSpCas9
Ampicillin
A / 10 B-SpCas9-HF1
Ampicillin
A / 11 B-HypaSpCas9
Ampicillin
A / 12 B-HypaR-SpCas9
Ampicillin
B / 1 B-HeFSpCas9
Ampicillin
B / 2 eSpCas9-plus
Ampicillin
B / 3 SpCas9-HF1-plus
Ampicillin
B / 4 B-Sniper SpCas9
Ampicillin
B / 5 B-HiFi SpCas9
Ampicillin
B / 6 pET-FLAG-eSpCas9
Kanamycin
Data calculated @ 2024-04-15

Kit Plate Map - #1000000235

Plate #1

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B
C
D
E
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