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CRISPR Background

CRISPR stands for clustered regularly interspaced short palindromic repeat; CRISPR-Cas (CRISPR-associated) systems are used for genome editing in various host organisms.

With their highly flexible but specific targeting, CRISPR-Cas systems can be manipulated and redirected to become powerful tools for genome editing. CRISPR-Cas technology permits targeted gene cleavage and gene editing in a variety of eukaryotic cells, and because the endonuclease cleavage specificity in CRISPR-Cas systems is guided by RNA sequences, editing can be directed to virtually any genomic locus by engineering the guide RNA sequence and delivering it along with the Cas endonuclease to your target cell.

TAL and CRISPR directly edit the genome by a combined cleavage and repair mechanism to impart permanent genomic change (deletion or frameshift mutation), and the resulting gene knockouts are very efficient. RNAi technology, on the other hand, is an indirect method in either down-regulating or shutting down a gene completely through direct interaction with RNA (coding or noncoding). Even in the case for stably expressed miRNA or shRNA systems, it may be difficult to effect complete penetrance (i.e., shRNA:mRNA ratio) since knock-down levels are dependent on the activity of the promoter (related to integration location).

As a simple two-component system that includes the Cas9 endonuclease and a noncoding guide RNA (gRNA), the engineered Type II CRISPR/Cas system can be leveraged to cleave genomic DNA at a predefined target sequence of interest. The gRNA has two molecular components: a target-complementary CRISPR RNA (crRNA) and an auxiliary trans-activating crRNA (tracrRNA). Both the gRNA and the PAM (NGG) motif guide the Cas9 nuclease to a specific genomic sequence to form a complex, followed by local strand separation (R-loop), at which the Cas9 nuclease creates a double-stranded DNA break (DSB) 3 nucleotides upstream from the PAM site. As a result, you may bring new functionality to the gene of interest via mutations, create knockouts, or introduce nonnative or synthetic genomic sequences to investigate novel applications. 

CRISPR also allows for non-editing application flexibility such as gene regulation or RNAi-related studies. The Cas9 nuclease may be tethered to different functional domains (activators or repressors) or the gRNA may be designed to directly cleave miRNA. 

There are 3 components:

  • The Cas nuclease Cas9 (a double-stranded DNA endonuclease)
  • A target-complementary crRNA
  • An auxiliary tracrRNA

The crRNA and the tracrRNA of the Invitrogen™ GeneArt™ CRISPR Nuclease Vector are expressed together as a guide RNA that mimics the natural crRNA-tracrRNA chimera in bacterial systems. 

 

Clonal isolation and a combined cleavage analysis and sequence verification of the edited clone is advisable.

Both HDR (homology directed repair) and NHEJ (non-homologous end joining) are cellular mechanisms through which double-stranded DNA lesions are repaired. When a repair template is not present, NHEJ occurs to ligate double-stranded breaks, leaving behind insertion/deletion (indel) mutations. HDR is an alternative repair pathway in which a repair template is used to copy the sequence to the double-stranded break. You can, therefore, introduce specific nucleotide changes or DNA fragments into your target gene by using HDR with a repair template.

All of them may work, but for better efficiency, a longer homology arm is better (at least 500 bp (or more) on either side of the exogenous DNA). The homology length is dependent on the size of the fragment and will need to be tested. ssDNA may be error-prone or choose NHEJ. We offer the Invitrogen™ GeneArt Strings™ dsDNA fragments (1–3 kb) to assist with this type of application.

Create a double-stranded DNA break using the GeneArt CRISPR Nuclease Vector, while simultaneously transfecting your plasmid-based donor repair template. Your donor repair template plasmid will contain the sequence you wish to introduce that is flanked by at least 500 bp (or more) of sequence, which results in efficient homologous recombination of your sequence.

HDR efficiency is very low, on average less than 2%.

Carefully designed crRNA target oligos and avoiding homology with other regions in the genome are critical for minimizing off-target effects.

Yes, this should be possible using CRISPR technology combined with HDR.

The first few exons would be best (closer to the promoter, resulting in premature transcript termination). Since the gRNA efficiency depends on the accessibility of the locus as well as the chromatin structure at that location, it is advisable to design and test a few target sites. Non–CRISPR-related mutations may be identified using gDNA isolated from non-CRISPR–treated cells as a control and performing a Invitrogen™ GeneArt™ Genomic Cleavage Detection Assay. Standard western blot analysis is a good measure for the verification of protein levels.

The gRNA oligo design strategy in the Invitrogen™ GeneArt™ CRISPR Nuclease User Guide describes how you can design the guide RNA to target the locus in which the neomycin cassette should be inserted. The cassette (neomycin) can be inserted via HDR, in which case the neomycin cassette should contain locus specific homology arms.

A single guide RNA (gRNA) is all that is required for targeting, but we do recommend that 2 or 3 gRNAs are tested against each locus being targeted for cleavage. Testing multiple gRNAs increases the chances of finding a gRNA with high editing efficiency. 

Yes, for all inquiries contact custom.services@lifetech.com and we can assist you with your project design and quote.

We have only tested these in mammalian systems (human and mouse cells).

Yes, it is possible but our system is not for prokaryotes, and has only been optimized for mammalian systems. Please also consult our CRISPR custom services for further inquiries (custom.services@lifetech.com).

Cas9 is transiently expressed and will therefore disappear over time with successive cell divisions.

With mRNA, we start seeing knockdown as early as 24 hours, with more efficiency observed after 48–72 hours.

Cleavage is precise, and, after binding of the Cas9 and gRNA complex to the target genomic sequence, the nuclease activity occurs 3 bases upstream of the PAM (NGG) site.

Since cleavage efficiency at a particular locus depends on the accessibility of the locus, chromatin state, and sequence, it is advisable to test multiple different loci/regions within a gene of interest. With CRISPR-Cas9–mediated genome editing, for each target of interest the user needs only to change the 19–20 bp target-specific oligo. After the cell lines have been screened and the sequence/locus with the highest cleavage efficiency has been identified, the biologically relevant mutations can be precisely created with high-specificity Invitrogen™ GeneArt™ TALs.

An indel refers to the genomic insertion or deletion of bases, which are incorporated during either cellular NHEJ or HD repair mechanisms.

Our CRISPR products are optimized for mammalian systems, and have not been optimized for plant systems. However, we know researchers are doing that kind of work. While we have not tested our CRISPR products for plants, our new protein format would be ideal since it does not need to be translated and transcribed in the cell, so no plant-specific promoters required.

For transfecting of the Cas9 protein, we would recommend using the Neon™ transfection system or Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent. For transfection of mRNA, we would recommend using Lipofectamine™ MessengerMAX™ Transfection Reagent. For transfection of CRISPR vectors, we would recommend using Lipofectamine™ 3000 Transfection reagent.

The only complete way to confirm that there are no off-target effects is to sequence the entire genome of your cell. Alternatively, a less thorough means of checking for off-target editing is to perform targeted sequencing of sequences with the highest probability of off-target effects (i.e., most similar to your CRISPR target region).

GeneArt CRISPR Nuclease Vectors

We recommend using the GeneArt CRISPR Nuclease Vector system when working with mammalian cells and in scenarios where there are no promoter constraints. We recommend using the GeneArt CRISPR Nuclease mRNA system when working with difficult-to-transfect cell lines, microinjections and for multiplexing.

This system allows you to edit and engineer the genomic locus of your choice in a sequence-specific manner from a single plasmid. We offer two plasmids that can be used with these kits, with either the OFP reporter or CD4 reporter, for ease of selection and enrichment by either FACs or Invitrogen™ Dynabeads™ technology. Both kits include all-in-one vectors that co-express both the noncoding guide RNA (including crRNA and tracrRNA) and the Cas9 endonuclease.

5’-nonphosphorylated desalted oligonucleotides are fine. However, HPLC or higher purity will increase the cloning efficiency of your double-stranded oligonucleotide into the GeneArt CRISPR Nuclease vector.

Either nuclease-free water or TE buffer is fine, but please anneal using the appropriate Oligonucleotide Annealing Buffer for your annealing reaction. Subsequently, please serially dilute your primers in a final concentration of 1X Oligonucleotide Annealing Buffer.

Our vectors contain a CMV promoter to drive expression of the Cas9 endonuclease.

The OFP reporter allows for fluorescence-based tracking of transfection efficiency, as well as FACS-based sorting/enrichment of Cas9- and CRISPR-expressing cells.

Populations of cells transfected with GeneArt CRISPR nuclease vectors containing CD4 reporters may be enriched using FACs or using Invitrogen™ Dynabeads™ CD4 magnetic beads.

Start off by designing two single-stranded DNA oligonucleotides (24–25 bp), one encoding the target-specific crRNA (forward-strand oligonucleotide) and the other its complement (reverse complement–strand oligonucleotide). You can generate a double-stranded oligonucleotide suitable for cloning into the linearized vector provided in the kit by simply annealing the complementary oligonucleotides. Here are some guidelines:

  • Length: Choose a target sequence ranging from 19 to 20 nucleotides in length that is adjacent to an NGG proto-spacer adjacent motif (PAM) sequence on the 3´ end of the target sequence. The 5´ G required for transcription initiation from the U6 Pol III promoter is already included in the vector overhangs and does not need to be included in the target sequence. Note: Do not include the PAM sequence in the oligonucelotide primers.
  • Homology: Make sure that the target sequence does not contain significant homology to other genes, as this can increase off-target effects. Recently published work has shown that gRNA-Cas9 complexes can potentially tolerate 1–3 or more mismatches, depending on their location in the gRNA. Refer to published articles for more insights into choosing a target sequence (Fu et al., 2013; Mali et al., 2013).
  • Orientation: You may choose a target sequence encoding either the sense sequence of the target locus or the antisense sequence. Thus, you can generate CRISPR RNA in two possible orientations, provided that it meets the PAM requirements on the 3´ end.

Yes, we provide kits both with or without competent cells. However, the vectors were optimized using Invitrogen™ One Shot™ TOP10 chemically competent E. coli, designed for high-efficiency cloning and plasmid propagation and stable replication of high-copy number plasmids. Please note that using competent cells of different genotype may lower cloning efficiency and can also result in a higher proportion of vectors without insert.

Confirm the identity of the ds oligonucleotide insert in positive transformants by sequencing. Analyze each CRISPR nuclease construct to verify:

  • That the ds oligonucleotide insert is present, and in the correct orientation
  • That the ds oligonucleotide insert has the correct sequence 

Note: Restriction analysis is not recommended due to the small size of the ds oligonucleotide insert.

We recommend using high-quality DNA prepared with the Invitrogen™ PureLink™ HiPure Plasmid Miniprep Kit. Ensure that DNA is free of contamination with phenol and sodium chloride.

The method of transfection varies based on cell type. For high-efficiency transfection in a broad range of mammalian cell lines, we recommend using the cationic lipid–based Invitrogen™ Lipofectamine™ 3000 Reagent.

All reagents are guaranteed to be stable for 6 months when properly stored. The vector, buffers, control oligo, plasmid, water, and sequencing primers should be stored at –20°C. 

gRNA expression is driven by the Pol III U6 promoter.

OFP has a peak excitation of 548 nm, and emission of 560 nm. We recommend a 488 nm laser for efficient excitation. Standard 530/30, 647/26, and 603/48 emission filters are recommended for detection.

Cleavage efficiency can be detected using the GeneArt Genomic Cleavage Detection Assay. This assay relies on mismatch detection endonucleases to detect insertions and deletions (indels) generated during cellular NHEJ repair.

With the CRISPR-Cas9 vector, transfect a DNA repair template that contains high homology to the sequence of interest along with the desired sequence you would like to introduce into the DNA. By doing so HDR can occur, and your specific edits (mutation, insertion, etc.) can be incorporated into the genome. 

PAM stands for the protospacer adjacent motif. It is necessary for Cas9 to bind to the DNA successfully. The PAM sequence for the Streptococcus pyogenes Cas9 in the GeneArt CRISPR kits is NGG. 

No, the PAM sequence is unique to the bacterial species that was used to create the Cas9. In the GeneArt kits, we derived Cas9 from Streptococcus pyogenes.

Yes, if you use the current GeneArt CRISPR nuclease vectors the respective Limited-Use Label Licenses (LULLs) will apply.

GeneArt CRISPR Nuclease mRNA System

The kit contains a ready-to-transfect wild type Cas9 mRNA for performing CRISPR-Cas9–mediated genome editing. The Cas9 mRNA can be used in experiments through two methods:

 Ready-to-transfect format: Cas9 mRNA is co-transfected directly with custom Invitrogen™ GeneArt™ CRISPR U6 Strings™ DNA or other synthetic gRNA expression cassettes.

Complete RNA format: Cas9 mRNA is co-transfected with in vitro transcribed gRNA. In vitro transcribed gRNA can be generated from Invitrogen™ GeneArt™ CRISPR T7 Strings™ DNA or other custom templates. 

Following transfection, the Cas9 protein (generated by the mRNA) is directed by the crRNA sequence of the gRNA to the encoded genomic locus to perform the desired genome editing.

In most cell types tested, this complete RNA format exhibits higher cleavage efficiency than the plasmid format. Additionally, the Cas9 mRNA format circumvents the need for cloning, has a smaller payload size, allows Cas9-to-gRNA dosage optimization, flexibility with multiplexing, and does not have any promoter constraints.

The CRISPR mRNA system contains ready-to-transfect wild type Cas9 mRNA that circumvents the need for a cell type–specific promoter.

Yes, the GeneArt CRISPR Strings DNA is offered for custom order with either U6 or T7 promoters.

Yes, if you have your own system to make a gRNA with a S. pyogenes TRACR sequence, it is not necessary to order GeneArt CRISPR Strings DNA. 

Co-transfect the GeneArt CRISPR U6 Strings DNA with GeneArt CRISPR Nuclease mRNA using Invitrogen™ Lipofectamine™ MessengerMAX™ Reagent. Analyze samples for gene modification 48–72 hours post-transfection using the GeneArt Genomic Cleavage Detection Assay or by sequencing.

We recommend Lipofectamine MessengerMAX reagent. 

Create multiple gRNAs targeting the targets of your choice, followed by co-transfection with GeneArt™ CRISPR Nuclease mRNA or GeneArt™ Platinum™ Cas9 Nuclease. To make the gRNAs for Cas9 mRNA, use GeneArt™ CRISPR Strings DNA, U6 or IVT gRNAs (generated using either GeneArt™ CRISPR Strings DNA, T7 or the GeneArt™ Precision gRNA Synthesis Kit). For the Cas9 protein, use  IVT gRNAs (generated using either GeneArt™ CRISPR Strings DNA, T7 or the GeneArt™ Precision gRNA Synthesis Kit).

We have tried our mRNA nuclease CRISPR system in multiple cell lines including: 293, HeLa, U2OS, HCT116, mouse neuro-2a, mouse ES cells, iPS cells, K562, Jurkat cells, CHO cells, and A549.

We recommend starting at a ratio of 0.5 µg of Cas9 mRNA and 50 ng of each IVT gRNA per well in a 24-well format. You should determine the optimal ratio for your particular cell line via a dose-response study.

Yes, the Neon system does work for multiple gRNAs transfected at the same time.

GeneArt™ CRISPR nuclease mRNA is a ready-to-transfect, mammalian codon-optimized Streptococcus pyogenes Cas9 nuclease mRNA that is properly capped and polyA-tailed for stability and high expression. The smaller payload of GeneArt™ CRISPR nuclease mRNA allows for single or multiplex gRNA delivery for CRISPR-mediated genome editing applications. High genome editing efficiency can be achieved when both CRISPR mRNA and gRNA are delivered with Lipofectamine™ MessengerMAX™ Transfection Reagent.

We recommend using a positive gRNA control with the GeneArt™ CRISPR nuclease mRNA to ensure that your transfection reagent was efficiently delivered to your cells. We have a functionally validated in vitro transcribed HPRT gRNA control available from our Custom Services group (send an email to custom.services@lifetech.com). Either the GeneArt™ Genomic Cleavage Detection Kit (Cat. No. A24372) or GeneArt™Genomic Cleavage Selection Kit (Cat. No. A27663) may be used to confirm genomic cleavage activity by Cas9 nuclease.

Lipofectamine™ MessengerMAX™ Transfection Reagent is especially formulated for the delivery of both mRNA and gRNAs for all cell types (easy or difficult-to-transfect, primary, and stem cells).

When the GeneArt™ CRISPR nuclease mRNA is used with the GeneArt™ Genomic Cleavage Selection Kit (Cat. No. A27663), both Cas9/gRNA ribonucleoprotein function and edited cells can be simultaneously confirmed by either OFP or CD4 selection.

GeneArt™ Platinum Cas9 Nuclease

The buffer composition for the Platinum Cas9 protein is as follows: 

10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.6 mM TCEP, 50% glycerol.

Please note that we can make custom formulations if necessary.

We recommend using the Neon™ Transfection System or the Lipofectamine™ CRSIPRMAX™ Cas9 Transfection Reagent. 

The molar ratio of IVT gRNA to Cas9 protein should be approximately 1.2:1. 

Guide RNA (gRNA) Synthesis

No other kits are required for this process. All necessary components are included in the kit.

The Precision gRNA Synthesis Kit (Cat. No. A29377) includes primers for synthesis of gRNA targeting safe harbor locus HPRT. We also have a fully processed, fully validated HPRT gRNA with GCD primers for confirmation of cleavage available from our custom services team.

Invitrogen™ GeneArt™ Genomic Cleavage Selection Kit

The GeneArt Genomic Cleavage Selection Kit can be used to:

  • Screen for functionality of your engineered nucleases as early as 24 hours post-transfection using fluorescence microscopy
  • Enrich for modified cells using fluorescence-activated cell sorting (FACS) or Dynabeads CD4 Magnetic Beads

The vector in the kit has an OFP gene that is interrupted by the insertion of a cloning site for the target sequence of the programmable nuclease. The upstream sequence coding for the N-terminal portion of the OFP gene contains a region complementary to the 5’ end of the C-terminal region of the OFP gene. A stop codon after the N-terminal OFP sequence ensures no expression of the reporter prior to nuclease activity. The CD4 gene is out of frame for expression when the OFP gene is interrupted by the cloning site. Double-stranded breaks cause the complementary strands of the end sequences of the OFP gene to recombine, and OFP expression is restored. The CD4 gene is now in frame for expression and can be screened for via FACs analysis or Dynabeads magnetic bead selection.

Yes, the GeneArt Genomic Cleavage Selection Kit can be used with either TALs or CRISPR constructs.

Please see the comparison table below:

GeneArt Genomic Cleavage Selection Kit GeneArt Genomic Cleavage Detection Kit
Fast, live detection Requires cells to be lysed
Visual indication (fluorescence) Quantifiable results
Proves editing tool works Negative result does not indicate whether editing tool works or not
Allows for clone enrichment No enrichment capabilities

It is possible to verify cleavage as early as 24 hours post-transfection by checking for OFP expression of the transfected cells under the microscope. The percentage of OFP-positive cells indicates the cleavage activity of TAL or CRISPR-Cas9.

The GeneArt genomic cleavage selection vector also contains the membrane protein CD4 coding gene that is fused with OFP through the T2A self-cleavage peptide, allowing nuclease-modified cells to be enriched through cell sorting or CD4 antibody–conjugated Dynabeads. This cleavage selection vector allows simple, rapid evaluation of the functionality of the programmable nuclease, and direct enrichment of the genome-modified cells.

OFP and CD4 expression are considered an estimation rather than absolute quantification of genomic cleavage. We recommend using our GeneArt Genomic Cleavage Detection Kit to verify cleavage on endogenous genomic locus.

OFP has peak excitation of 548 nm and emission of 560 nm. A 488 nm laser is recommended for efficient excitation. Standard 530/30, 574/26 and 603/48 emission filters are recommended for detection.

Invitrogen™ GeneArt™ Genomic Cleavage Detection Kit

The GeneArt Genomic Cleavage Detection Kit provides a simple, reliable, and rapid method for the detection of locus-specific double-strand break formation. This technique relies on mismatch detection endonucleases to detect genomic insertions or deletions (indels) incorporated during cellular NHEJ repair mechanisms.

The assay uses genomic DNA extracted from cells transfected with constructs expressing engineered nucleases. Following cleavage, indels are created by the cellular repair mechanisms. Loci where the gene-specific double-stranded breaks occur are amplified by PCR. The PCR product is denatured and reannealed so that mismatches are generated as strands with an indel reannealed to strands with no indel or a different indel. The mismatches are subsequently detected and cleaved by Detection Enzyme, and then the resultant bands are analyzed by gel electrophoresis and band densitometry.

Genomic DNA at the locus being investigated must be PCR amplified prior to detection. Follow these recommended guidelines to ensure optimal amplification and subsequent detection:

  • For best results, use primers with Tm >55°C.
  • Design primers that are 18–22 bp in length and have 45–60% GC content.
  • For efficient amplification, design primers to yield amplicon lengths between 400 and 500 bp.
  • Design primers so that the potential cleavage site is not in the center of the amplicon and that the detection reaction will yield two distinct product bands.