User Instructions:Transfection-ready CRISPR/Cas9 Reagents. Target DNA. NHEJ repair pathway. Nucleotide deletion. Nucleotide insertion Gene disruption

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1 User Instructions:Transfection-ready CRISPR/Cas9 Reagents Background Introduction to CRISPR/Cas9 genome editing In bacteria and archaea, clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) constitute an adaptive immune system against phage and other invading genetic elements. The system identifies repeat infections by integrating short sequences of invading genomes into a specific region of the host genome (the CRISPR locus) between short palindromic DNA repeats. The entire locus is transcribed, and transcripts are then processed into small CRISPR RNAs (crrnas) that are employed by Cas proteins to target invaders sequencespecifically upon a reoccurring infection. s SpCas9 protein Target DNA PAM sequence Generate double-strand break Among many identified CRISPR-Cas systems, the Streptococcus pyogenes type II CRISPR/Cas9 system has been well-characterized and engineered for genome editing in mammalian cells. In S. pyogenes, only three components are required for targeted DNA cleavage: the Cas9 protein, the mature crrna and the tracrrna. In 2012, a simplified two-component system was developed by combining tracrrna and crrna into a synthetic single guide RNA (s). s-programmed Cas9 can be effective in guiding targeted gene alterations. Inside a cell, the Cas9 protein combines with the s to form an active ribonucleoprotein (RNP) complex. The RNP complex scans the cellular genome for PAM (protospacer adjacent motif) sequences and unwinds double-stranded DNA near a PAM site, allowing the s to base NHEJ repair pathway Nucleotide deletion Nucleotide insertion Gene disruption Nucleotide to bemutated Corrected nucleotide HDR repair pathway Donor DNA Precise repair Figure 1. CRISPR-induced NHEJ and HDR. pair with now single-stranded DNA. The Cas9 protein then either cleaves the sequence close to the PAM site or the RNP complex moves on to the next PAM, depending on the base-pairing between s and target DNA. For SpCas9, the PAM sequence is NGG, while other Cas proteins use different PAM sequences. SpCas9 nuclease cuts both DNA strands to generate a blunt-ended double-strand break (DSB) at 3 bp 5 of the PAM. Most DSBs are then repaired by either the non-homologous end-joining (NHEJ) pathway or the homology-directed repair (HDR) pathway. In mammalian cells, the NHEJ pathway is predominant and error-prone, often resulting in small insertions or deletions (indels) at the break site. NHEJ can be exploited to generate gene knockouts, as indels taking place within a coding exon can introduce nonsense mutations. Furthermore, NHEJ can be exploited to mediate deletion of a chromosomal DNA fragment flanked by two DSBs. Unlike HDR, NHEJ is active in both dividing and non-dividing cells. HDR is an alternative major DNA repair pathway. Although HDR typically has lower or more variable frequency than NHEJ, it can be leveraged to make precise gene modifications in the presence of an exogenous repair template. The repair template can be single-stranded(ssdna) or double-stranded DNA (dsdna) with a desired insert flanked by homology arms complementary to the genomic regions adjacent to the cutting site. ssdna repair templates provide a simple and cost-effective method for making small sequence changes efficiently, such as insertion of tags and point mutation. Unlike NHEJ, HDR is generally active only in dividing cells, and its efficiency can vary widely depending on the cell type and state, as well as the genomic locus. Page 1 of 6

2 VectorBuilder Creation of isogenic cell lines using CRISPR/Cas9 Introducing CRISPR/Cas9 components into in vitro cultured cells always results in a heterogeneous population of cells with different editing outcomes among individual cells. It's therefore essential to isolate correctly targeted and genetically homogeneous cell lines to facilitate downstream experiments. To obtain a clonal population with a homogeneous editing outcome, clones from the primary transfected pool of cells need to be screened. This requires at least two rounds of screening. At primary screen, T7E1 mismatch cleavage assay is recommended to perform in a fraction of transfected cells to determine the remaining transfected cells containing an edit. Next, the polyclonal cell culture is further processed to isolate monoclones using limiting dilution technique. A secondary screen is performed to identify the expanded clones containing the desired edit. Sanger sequencing is recommended for ensuring the sequence change is desired. target site Exon Step 1 In silico design of CRISPR targeting Cas9 5' HA Step 2 Reagent preparation New sequence 3' HA dsdna or ssdna Cas9 and co-expressing plasmid Repair template (only for HDR applications) Repair template (optional) Plasmid DNA Step 3 Transfection of Cas9 and with or without repair template Lipofection or electroporation T7E1 mismatch cleavage assay Genotype Isolate clonal lines Step 4 Clonal expansion and validation Expand Genotype Sanger sequencing * Figure 2. Overall workflow for creating isogenic cell lines using CRISPR/Cas9. Page 2 of 6

3 Selection of CRISPR/Cas9 reagents The CRISPR components can be introduced into mammalian cells in a few ways, including expression plasmid, Cas9 mrna/, preformed Cas9- RNP complex, and viral vectors. Each method has its own set of advantages and limitations. Transfection of plasmid DNA is a simple and low-cost approach. Once inside the cells, plasmid DNA produces high-level expression of Cas9 protein and over several days, potentially leading to more complete modifications in addition to a higher degree of off-target cutting events. In addition, the risk of random integration of plasmid DNA into the host genome and choice of promoters for different cell types should also be considered. Delivery of Cas9 mrna/ enables editing to occur more rapidly, as no transcription is required. But the editing activity drops off quickly as the transcripts get degraded inside their host cells. Using Cas9 mrna/ eliminates the risk of insertional mutagenesis and reduces the possibility of off-target cutting events. Delivery of preformed RNP complex results in the most rapid pulse of editing activity as neither transcription nor translation is necessary. Inside the cells, without continuous expression of Cas9, Cas9 protein turns over rapidly, leading to the lowest level of off-targeting compared to other methods. Using preformed RNP complexes with repair template facilitates higher HDR rates. Lentivirus, adenovirus, and adeno-associated virus (AAV) can be used to introduce CRISPR/Cas9 components. These viral methods enable efficient genome editing in hard-to-transfect cells due to their ability to infect both dividing and non-dividing cells, as many cell types resistant to transfection by chemical and liposome methods are non-dividing cells. Delivery tools Traditional non-viral techniques for delivering macromolecules into in vitro cultured cells include transfection and electroporation. Transfection techniques make use of chemical reagents (either lipids, polymers or some combinations thereof) to overcome the cell-membrane barrier. The principle consists of the interaction of negatively charged nucleic acids with positively charged transfection reagents, enabling the nucleic acid to come in contact with the negatively charged cell membrane and incorporating the nucleic acid into the cell by endocytosis and later release it in the cytoplasm. Lipofection is the most commonly used chemical transfection method due to its ability to deliver nucleic acids and proteins into a wide range of cell types with high efficiency, and at low cost. Electroporation delivers editing materials by giving an electrical current to cells. Electrical pulse induces temporary nanometer-sized pores in the cell membrane for nucleic acid and protein entry. This method is less dependent on the cell type and can efficiently transfer editing materials into difficult-to-transfect cells, but it requires costly hardware and consumables. Viral delivery techniques don't require chemical transfection reagents, electroporation hardware and consumables. The viruses infect mammalian cells by receptor-mediated endocytosis followed by releasing of their viral genomes. Virus/cell interaction is very efficient. Therefore, viral delivery can work under more complicated in vivo conditions than non-viral delivery techniques. Page 3 of 6

4 Content The components of CRISPR/Cas9 system can be produced in multiple ways for various genome editing applications. The following table shows available CRISPR/Cas9 reagents for common genome editing applications in mammalian cell lines. Common CRISPR/Cas9-mediated Genome Editing Applications in Mammalian Cells Available CRISPR/Cas9 Reagents Cas9/ plasmid Gene disruption by NHEJ Cas9 mrna + Cas9 protein + Cas9/ virus (lentivirus, adenovirus or AAV virus) Two or more Cas9/ plasmids Multiple gene disruptions by NHEJ Cas9 mrna + two or more s Cas9 protein + two or more s Two or more Cas9/ viruses (lentivirus, adenovirus or AAV virus) Cas9/dual- plasmid Cas9 mrna Direct chromosomal DNA deletion by paired-ko strategy Cas9 protein Cas9/dual- virus (lentivirus or adenovirus) Two Cas9/ AAV virus Cas9/ plasmid + ssdna donor Introduce a small DNA sequence (point mutation, small tag insertion) Cas9 mrna + + ssdna donor Cas9 protein + + ssdna donor Generate a conditional allele by inserting two loxps Cas9/dual- plasmid + ssdna donor 1 + ssdna donor 2 Cas9 mrna ssdna donor 1 + ssdna donor 2 Cas9 protein ssdna donor 1 + ssdna donor 2 Cas9/ plasmid + plasmid donor Introduce a large DNA sequence by HDR Cas9 mrna + + plasmid donor Cas9 protein + + plasmid donor Generate stable Cas9-expressing cell lines* Cas9 plasmid Cas9 lentivirus * With cell lines that express Cas9 continuously, functional RNP complexes can form rapidly at the time of introducing ss into the cells, leading to higher efficiency of cleavage and higher HDR rates. Page 4 of 6

5 The following table lists and illustrates the product specifications, shipping and storage conditions for transfection-ready CRISPR/Cas9 reagents. Reagents Composition Quantity Shipping/Storage Transfection-ready Cas9/ plasmid >1 ug/ul in 1x TE buffer, endo-free, sterile 150 ul RT/-20 C Transfection-ready Cas9/dual- plasmid Transfection-ready Cas9 mrna (Cat # R00112) Transfection-ready Cas9(D10A) nickase mrna (Cat # R00212) Transfection-ready Cas9 nuclease (Cat # R00360) >1 ug/ul in 1x TE buffer, endo-free, sterile 150 ul RT/-20 C >/ul in deionized and nuclease-free water 25 ul Dry ice/-80 C >/ul in deionized and nuclease-free water 25 ul Dry ice/-80 C 3 ug/ul 20 ul Dry ice/-20 C Transfection-ready >/ul in deionized and nuclease-free water 25 ul Dry ice/-80 C Donor oligo (for transfection) Dry powder 2 nmol RT/RT Transfection-ready donor plasmid (linearized) Cas9/ lentivirus >500 ug/ul in 1x TE buffer, endo-free, sterile 50 ul RT/-20 C 8 >10 TU/ml in HBSS buffer 1 ml Dry ice/-80 C Cas9/ adenovirus Cas9/ AAV virus Cas9/dual- lentivirus Cas9/dual- adenovirus 10 >10 PFU/ml in HBSS buffer 1 ml Dry ice/-80 C 11 >10 GC/ml in Tris buffer 1 ml Dry ice/-80 C 8 >10 TU/ml in HBSS buffer 1 ml Dry ice/-80 C 10 >10 PFU/ml in HBSS buffer 1 ml Dry ice/-80 C Transfection-ready Cas9 plasmid >1 ug/ul in 1x TE buffer, endo-free, sterile 150 ul RT/-20 C Cas9 lentivirus 8 >10 TU/ml in HBSS buffer 1 ml Dry ice/-80 C Protocol for transfection of CRISPR/Cas9 components into adherent cells : 1. Day before transduction (Day 0) Plate target cell in appropriate medium so that they will be % confluent at the time of transfection. Incubate hours at 37 C in a humidified 5% CO 2 incubator. For example, when using 293T cells, we recommend plating cells per well in a 24-well pate. 2. Day of transduction (Day 1) Prepare transfection complex for plasmid DNA transfection as follows : For NHEJ applications, transfection of Cas9/ plasmid can be performed using Lipofectamine 2000, with of plasmid per well of a 24-well plate, according to the manufacturer's instructions. If you are transfecting more than one plasmid, mix them at equimolar ratios and use no more than of total DNA. For HDR applications, co-transfection of Cas9/ plasmid and repair template can be performed using Lipofectamine 3000 according to the manufacturer's instructions. Mix the components as follows: For the co-transfection of donor plasmid with Cas9/ plasmid: Cas9/ plasmid Linearized donor plasmid For the co-transfection of donor oligo with Cas9/ plasmid: Cas9/ plasmid ssdna (10 um) 1 ul Page 5 of 6

6 Prepare transfection complex for Cas9 mrna transfection as follows: For NHEJ applications, co-transfection of Cas9 mrna and can be performed using either Lipofectamine 3000, MessengerMAX or RNAiMAX according to the manufacturer s instructions. Mix the components in an RNase-free microcentrifuge tube as follows: Cas9 mrna ng For HDR applications, co-transfection of Cas9 mrna, and repair template can beperformed using either Lipofectamine 3000, MessengerMAX or RNAiMAX according to themanufacturer s instructions. Mix the components in an RNase-free microcentrifuge tube asfollows: For the co-transfection of donor plasmid with Cas9 mrna/: Cas9 mrna ng Linearized donor plasmid For the co-transfection of donor oligo with Cas9 mrna/: Cas9 mrna ng ssdna (10 um) 1 ul Prepare transfection complex for Cas9 protein transfection as follows: For NHEJ applications, preformed RNP complex can be transfected using either Lipofectamine3000, MessengerMAX, or RNAiMAX according to the manufacturer s instructions. Form thernp complexes in an RNasefree microcentrifuge tube as follows: Cas9 protein Opti-MEM 120 ng 25 ul The molar ratio of to Cas9 protein should be kept at approximately 1 to 1.2:1. Gently mix the reaction and incubate at room temperature for 10 minute. In a separate RNase-free microcentrifuge tube, 2 ul of Lipofectamine 3000, MessengerMAX, or RNAiMAX is added to 25 ul of Opti-MEM medium. Add the diluted transfection reagent to the RNP tube. Gently mix the liposome complexes and incubate at room temperature for minutes. For HDR applications, form RNP complexes as described above. Incubate 10 min at room temperature. Then add 1 ul of 10 um ssdna or linearized donor plasmid to the RNP tube. Proceed to the formation of liposome complex as described above. In each case, the entire solution is added to the cells in the 24-well plate. Swirl the plate gently tomix and incubate at 37 C in a humidified 5% CO incubator overnight Day 2 If Cas9/ plasmid contains a fluorescent mark (e.g., EGFP), check transfection efficiency after24 h by visualising the fluorescent cells under a fluorescence microscope. Typically, more than70% of cells are transfected. Remove the medium and replace with fresh complete culture medium If necessary. Incubate thecells for a total of h after transfection before passaging them for downstream clonalanalysis or harvesting for indel analysis. Page 6 of 6

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