We thank many previous members of our labs and the Bay Area genome editing community for their contributions to the development of these methods. We thank Ross Wilson for critically reading this manuscript.
Alexander Marson's research is supported by a gift from the Jake Aronov and a National Multiple Sclerosis Society grant (CA 1074-A-21). Alexander Marson holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund and is a Chan Zuckerberg Biohub Investigator. Jacob E. Corn's research is supported by the Li Ka Shing Foundation, the Heritage Medical Research Medical Institute, and the California Institute for Regenerative Medicine. Behnom Farboud and Barbara J. Meyer's research is funded in part by the NIGMS grant R01 GM030702 to Barbara J. Meyer, who is an investigator of the Howard Hughes Medical Institute. Erin Jarvis and Nipam H. Patel's research is funded in part by the NSF grant IOS-1257379 and Erin Jarvis acknowledges support from an NSF GRFP and a Philomathia Graduate Fellowship.

The authors Alexander Marson and Jacob E. Corn are co-founders of Spotlight Therapeutics. Jacob E. Corn is an advisor to Mission Therapeutics and his laboratory has received sponsored research support from AstraZeneca and Pfizer. Alexander Marson is an advisor to Juno Therapeutics and PACT Therapeutics, and his laboratory has received sponsored research support from Juno Therapeutics,&#160;Epinomics,&#160;and Sanofi. His laboratory has also applied for patents related to Cas9 RNP technology.

Establishing a robust genome editing protocol in a cell line or organism of interest requires the optimization and empirical testing of several key parameters, discussed in this section. Trying a few variations of the general approaches presented here is highly encouraged. The key limitation of this protocol is that applying these methods to other cells or organisms may lead to a different outcome depending on the species studied, and an experimental design that leads to a high-efficiency gene knockout may not promote DNA insertion. Thus, we recommend starting with the methods presented here and troubleshooting as described below.
Troubleshooting genome editing reagent quality: 
Generating or purchasing high-quality reagents is a critical step in any genome editing protocol. Cas9 protein can be purified in the lab or purchased commercially. Many protocols note a final concentration for Cas9 in RNP recipes, but the optimal gene editing activity will depend on the specific activity of any individual Cas9 protein preparation, which varies depending on the source. Once the protocol presented here is working, consider optimizing the amount of RNP used by titrating Cas9 levels to establish an optimal concentration: one that provides highly specific target DNA cleavage without unnecessary off-target cleavage caused by excessive Cas940.
Guide RNA purity and homogeneity can also be determinants of genome editing success22. Purchased sgRNAs or separate crRNA and tracrRNA components are generally high-quality reagents and a variety of chemical modifications are available to combat problems with RNA degradation or to imbue additional features to the RNP91. While chemically-modified gRNAs may not be necessary for standard genome editing experiments, some groups have observed much higher editing efficiencies with such reagents, so they may be worth trying after mastering the process and/or when gRNA degradation appears to be an issue22,91. In vitro transcription and subsequent gel purification is an inexpensive alternative, which may be sufficient for routine genome editing experiments17,21,49,50. Further, several approaches that are commonly applied to produce homogenous gRNA populations in vivo, including ribozyme- and tRNA-based excision of individual guides, may be extended to in vitro RNA preparation to generate cleaner products92.
Guide RNA and donor DNA design tips: 
Guide RNA selection is a critical factor in achieving highly efficient on-target editing while minimizing the chances of off-target cleavage. To aid in guide selection, several studies have used high-throughput screens coupled with next-generation sequencing to compile sequence features of successful guides47,79,93,94,95,96. These features have been used to develop predictive algorithms and online tools to assist in guide selection44,45,46,47,48. Such algorithms are grounded on screens using DNA-based systems for guide RNA expression. Guides are expressed using a Pol III promoter, and their expression is therefore prone to the limitations associated with Pol III transcription, such as premature termination when encountering tracks of uracil97,98,99. However, use of RNPs made with in vitro-synthesized guide RNAs bypasses those concerns and simplifies the constraints on guide design. A common feature that emerged from these algorithms and has been confirmed in numerous studies with highly effective genome editing, is the presence of a purine, particularly a guanine, at the 3&#8242; end of the guide&#39;s target-specific sequence. This guide feature has been very successful among organisms ranging from mammals to C. elegans, fruit flies, and zebrafish65,100,101. In addition, for C. elegans, designing guides with a GG dinucleotide at the 3&#8242; end of the guide&#39;s targeting region is an effective strategy for predicting highly effective guide RNAs65. Ideally, test multiple guides in parallel to determine which is most successful for a given application.
When attempting to introduce a DNA sequence into the genome, the design of the donor or template DNA is also crucial. Single-stranded oligonucleotide donors (ssODNs) are inserted more reliably than other typical repair templates, linear double-stranded and plasmid DNA54,55,102. At some loci, HDR efficiency can be improved with ssODNs that are complementary to the non-target or displaced DNA strand and possess homology arms that are asymmetric in length27,55. Since the repair template is being inserted at the cut site and includes the targeted sequence, steps must be taken to prevent Cas9 from cleaving the donor DNA before or after the genomic insertion. This is accomplished by making silent mutations to the PAM sequence or seed region, avoiding the recognition by Cas9 while retaining the function of the inserted gene21,103. Though even single nucleotide changes to the PAM are likely to abolish binding104, try to change at least four nucleotides to be safe.
Significance and future applications: 
Genome editing with CRISPR-Cas9 has emerged as a powerful method enabling facile genetic manipulation of any organism. Editing with the Cas9 RNP takes a bit more effort at first but is straightforward to use once reagents and protocols are established in a lab. Editing cells with pre-assembled RNP instead of plasmid DNA leads to higher overall editing efficiencies, including the difficult-to-achieve gene insertion via HDR, with fewer off-target effects24,25,26,27,29. Further, experimenters avoid problems with gene expression, RNA degradation, protein folding, and the association between gRNA and Cas9 molecules synthesized separately within the cell22,23. RNP editing also circumvents safety concerns about insertional mutagenesis and sustained expression that may arise when viral delivery methods are used clinically14. Because of these advantages, many scientists conducting pre-clinical, proof-of-concept experiments favor RNP editing for human therapeutic applications. Both in vivo and ex vivo RNP-based genome editing approaches are in development to treat or even cure a variety of conditions, from genetic diseases like Duchenne muscular dystrophy105 and sickle cell disease27 to HIV29 and cancer11. Interestingly, Cas9 RNP is increasingly employed as a delivery method for agricultural engineering because it enables &#39;DNA-free&#39; editing of plants33,34,36.

fThe CRISPR-Cas9 system allows scientists to alter targeted regions of any genome1. This quick and inexpensive technology has revolutionized basic research and promises to make a profound impact on the development of personalized disease therapies, precision agriculture, and beyond2. CRISPR editing is a democratizing tool and implementing the system in a new laboratory requires no particular expertise in genome engineering, just basic molecular biology skills. Researchers can now study previously intractable organisms with a few alternative means for genetic manipulation3,4. In the past five years alone, CRISPR genome editing has been used to engineer over 200 different vertebrates, invertebrate, plant, and microbial species.
Adapted from the CRISPR prokaryotic defense pathway, the core elements required for site-specific genome editing are the Cas9 protein, typically from S. pyogenes and codon-optimized with an added nuclear localization signal (NLS), and its specialized RNA guide5,6. Though not discussed here, other Cas9 orthologues or CRISPR endonucleases may also be used. The naturally occurring gRNA is composed of two separately transcribed pieces, the CRISPR RNA (crRNA) and the trans-activating crRNA (tracrRNA)7. These RNAs can be fused into a single transcript, known as the single-guide RNA (sgRNA)8. Most genome editors choose the streamlined sgRNA9, though the dual-guide is also used regularly10,11. Experimenters choose a 20-nucleotide (nt) genomic DNA target, ensuring that it lies next to a short licensing signature required for Cas9 recognition, called a protospacer adjacent motif (PAM), and design a gRNA that contains the complementary sequence12.
Once inside the cell, the RNP complex locates its genomic target, the gRNA base pairs with the complementary DNA strand, and then the enzyme cleaves both DNA strands to generate a double-strand break2. Cell repair machinery fixes the DSB by one of at least two routes: via the error-prone non-homologous end-joining (NHEJ) pathway or the homology-directed repair (HDR), which seamlessly incorporates DNA containing &#39;arms&#39; of homology to either side of the break. The former repair pathway typically leads to indel formation and consequent gene disruption, while the latter allows experimenters to insert or change DNA sequences1.
The editing efficiency and accuracy depend on the means by which Cas9 and gRNA enter into the cell. These components may be delivered to cultured cells, embryos, or organisms in the form of nucleic acids or as a preassembled RNP complex13,14,15. Common nucleic acid-based delivery methods include the viral transduction, transfection, or electroporation of mRNA or plasmid DNA. Cas9 protein and guide RNA are then produced within the cell and they associate to form a complex.
The direct delivery of RNP requires the separate purification of the Cas9 protein and guide RNA. This can be done in-house, or the protein and sgRNA can be purchased from one of several commercial vendors. Once acquired, the Cas9 and gRNA are mixed to form the enzymatically-competent RNP complex and introduced to cells by direct injection into fertilized eggs/embryos, lipid-based transfection16, or electroporation. The first report of RNP editing involved injection into C. elegans gonads17. Microinjection is still the preferred means of introducing RNP into embryos and whole organisms, though effective electroporation has been demonstrated in mouse18,19 and rat20 embryos. We describe protocols for directly injecting RNP into C. elegans gonads and P. hawaiensis embryos and recommend a specialized type of electroporation to deliver RNP when editing primary human cells. This method, nucleofection, involves optimized electroporation programs and cell type-specific solutions and allows the RNP to enter both the cytoplasm and the nucleus21.
Genome editing with RNP offers several distinct advantages. Because the protein and RNA components are pre-assembled, and quality can be ensured prior to delivery, RNP editing avoids many pitfalls associated with the nucleic acid-based delivery. Namely, there is no risk of Cas9-encoding DNA integration into the host genome, mRNA is never exposed for degradation, and it circumvents problems with in vivo gRNA or protein expression, folding, and association22,23. Further, using RNP leads to lower toxicity and far fewer off-target events than the plasmid-based expression, a result of the RNP&#39;s shorter half-life inside the cell24,25,26,27.
Finally, RNP editing demonstrably leads to high editing rates in a variety of human cell lines, primary cells such as fibroblasts, embryonic stem cells (ESCs), induced pluripotent stem cells (iSPCs), HSPCs, and T cells16,24,25,26,27,28,29; in invertebrates including C. elegans, P. hawaiensis, and fruit flies3,17,30; in vertebrate species like zebrafish, mice, and rats31,32; in plant species including Arabidopsis, tobacco, lettuce, rice, grapevine, apple, maize, and wheat33,34,35,36; and in Chlamydomonas, Penicillium, and Candida species37,38,39. The frequency of indel formation can be higher when using RNP compared to the plasmid delivery, and HDR-mediated DNA insertion can be easier to achieve25,27,29.
The protocol described here uses the Cas9 RNP and is an effective, readily adaptable technique that is straightforward to apply to a wide variety of biological systems40,41, especially in cells that are otherwise difficult to work with and in organisms without well-established systems for precise genetic manipulation. We start by describing how to design, obtain, and assemble the Cas9 RNP before covering its use across different model cell types and organisms. Hematopoietic stem/progenitor cells (HSPCs) and T cells are edited using the same method, nucleofection, so they are covered together in steps 2 and 3 of this protocol. Editing procedures for C. elegans are described in steps 4 and 5, and P. hawaiensis editing is covered in steps 6 and 7. Finally, since the success of a gene-editing experiment in any organism may be assessed by genotype sequencing, substeps describing possible analysis methods for all the cells and organisms described in the protocol are outlined in step 8.

<table><tbody><tr><td>&lt;strong&gt;Reagents/Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DNA oligonucleotides</td><td>Integrated DNA Technologies</td><td>-</td><td>IDT will provide custom DNA sequences, including those in Table 1</td></tr><tr><td>Guide RNAs</td><td>Synthego</td><td>-</td><td>Synthego will provide high-quality sgRNAs for S. pyogenes Cas9, including custom sgRNAs containing the targeting sequences included in Table 1</td></tr><tr><td>Purified Cas9 protein (EnGen&amp;nbsp;Cas9 NLS,&amp;nbsp;S. pyogenes)</td><td>New England Biosciences</td><td>M0646T</td><td>If possible, purifying Cas9 in-house or purchasing from local core facilities is a less expensive option</td></tr><tr><td>Normal peripheral blood CD34+ stem/progenitor cells</td><td>AllCells</td><td>PB032-2</td><td></td></tr><tr><td>StemSpan SFEM&amp;nbsp;</td><td>StemCell Technologies</td><td>09650</td><td></td></tr><tr><td>StemSpan CC110</td><td>StemCell Technologies</td><td>02697</td><td></td></tr><tr><td>P3 Primary Cell 4D-Nucleofector X Kit</td><td>Lonza</td><td>V4XP-3032</td><td></td></tr><tr><td>RPMI-1640 Medium, With sodium bicarbonate, without L-glutamine, liquid</td><td>Sigma</td><td>R0883-6X500ML</td><td></td></tr><tr><td>EasySep&amp;trade; Human T Cell Isolation Kit</td><td>Stemcell</td><td>17951</td><td></td></tr><tr><td>cell culture plate, 96 wells, round</td><td>Fisher Scientific</td><td>3799</td><td></td></tr><tr><td>CTS (Cell Therapy Systems) Dynabeads CD3/CD28</td><td>Life Tech</td><td>40203D&amp;nbsp;</td><td></td></tr><tr><td>Reombinant Human IL-2</td><td>UCSF Pharmacy</td><td>NA</td><td></td></tr><tr><td>SepMate-50 500-pack IVD</td><td>Stemcell Technologies</td><td>85460</td><td></td></tr><tr><td>OP50 &lt;em&gt;Escherichia coli&lt;/em&gt;</td><td>Caenorhabditis Genetics Center</td><td>OP-50</td><td>https://cgc.umn.edu/</td></tr><tr><td>Nematode Growth Media agar in petri dishes</td><td>-</td><td>-</td><td>See Stiernagle, T (ref. 59)</td></tr><tr><td>Standard borosilicate glass capillaries with filament:&amp;nbsp; 4 in (100 mm), 1/0.58 OD/ID</td><td>World Precision Instruments</td><td>1B100F-4</td><td></td></tr><tr><td>Single-barrel standard borosilicate glass capillaries: 6 in (152 mm), 2/1.12 OD/ID&amp;nbsp;</td><td>World Precision Instruments</td><td>1B200-6&amp;nbsp;</td><td></td></tr><tr><td>Cover glass; 24 &amp;times; 50 mm</td><td>Thermo Fisher Scientific</td><td>12-544E</td><td></td></tr><tr><td>Cover glass; 22 &amp;times; 22 mm</td><td>Thermo Fisher Scientific</td><td>12-518-105K</td><td></td></tr><tr><td>Apex LE agarose</td><td>Genesee Scientific</td><td>20-102</td><td></td></tr><tr><td>Halocarbon oil 700</td><td>Sigma-Aldrich&amp;nbsp;</td><td>H8898-100ML</td><td></td></tr><tr><td>pCFJ90 plasmid</td><td>Addgene</td><td>19327</td><td></td></tr><tr><td>Compressed nitrogen</td><td>-</td><td></td><td></td></tr><tr><td>60 mM culture dishes</td><td>BD</td><td></td><td></td></tr><tr><td>Capillary tubes with filament: 4 in (1.0 mm)&amp;nbsp;</td><td>World Precision Instruments</td><td>T2100F-4</td><td></td></tr><tr><td>Sylgard 184</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>Petri dishes (100 &amp;times; 15 mm)</td><td>-</td><td></td><td></td></tr><tr><td>Tungsten wire (0.005 in. diameter)</td><td>Ted Pella</td><td></td><td></td></tr><tr><td>Perfluoroalkoxy alkane (PFA)</td><td>-</td><td></td><td></td></tr><tr><td>Marine salt</td><td>-</td><td></td><td></td></tr><tr><td>9&quot; pasteur pipettes</td><td>-</td><td></td><td></td></tr><tr><td>Phenol red</td><td>-</td><td></td><td></td></tr><tr><td>Nuclease-free water</td><td>-</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4D Nucleofector</td><td>Lonza</td><td>AAF-1002X</td><td></td></tr><tr><td>MZ75&amp;nbsp;Stereomicroscope</td><td>Leica&amp;nbsp;</td><td>Out-of-production. Current model is the M80 Stereomicroscope</td><td></td></tr><tr><td>Axio Vert35 inverted phase contrast fluorescent microscope</td><td>Zeiss</td><td>Out-of-production. Current model is the Axio VertA.1</td><td></td></tr><tr><td>Laser-based micropipette puller (for C. elegans protocol)</td><td>Sutter Instrument&amp;nbsp;</td><td>FG-P2000</td><td></td></tr><tr><td>Picoliter Microinjector (for C. elegans protocol)</td><td>Warner Instruments</td><td>PLI-100A</td><td></td></tr><tr><td>Three-axis Joystick oil hydraulic micromanipulator</td><td>Narishige International</td><td>MO-202U</td><td></td></tr><tr><td>Coarse manipulator</td><td>Narishige International</td><td>MMN-1</td><td></td></tr><tr><td>Micropipette puller (for P. hawaiensis protocol)</td><td>Sutter Instrument&amp;nbsp;</td><td>P-80/PC</td><td></td></tr><tr><td>Microinjector (for P. hawaiensis protocol)</td><td>Narishige</td><td>IM300</td><td></td></tr><tr><td>Microloader pipette tips</td><td>Eppendorf</td><td>5242956003</td><td></td></tr><tr><td>NG-agar</td><td></td><td></td><td></td></tr></tbody></table>

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.

These experiments show how pre-assembled Cas9 RNP can be used to manipulate the genomes of primary cells and whole organisms. Researchers purify or purchase Cas9 protein and sgRNA, combine the two components to pre-form the complex, and introduce the RNP into their cells or organism of interest. After allowing enough time for editing to occur and for offspring of the next generation to be born (if applicable), check for phenotypes and/or collect cells for genotyping. Phenotypes may be observed via functional assays, expression assays, visualization (by eye or with microscopy), or other methods, depending on the experiment.
For example, HSPCs that have been edited to correct the &#946;-globin mutation that causes sickle cell disease can be differentiated into erythrocytes and assayed for the production of healthy or sickle hemoglobin27,87 (Figure 1A). T cells edited to knock out the high-affinity IL-2 receptor gene, CD25 (IL2RA), can be analyzed by surface staining and flow cytometry88, and functionally analyzed to detect a signaling response to IL-2 stimulation (Figure 1B). T cells can also be reprogrammed in many clinically important ways that require assessment of different phenotypes, including the efficacy of HIV infection89 and the in vivo antitumor efficacy of CAR-T cells11.
Using a co-CRISPR/co-conversion screening approach, C. elegans worms are edited simultaneously at two loci62. HDR at the dpy-10 reference gene using a ssODN repair template results in an easily-scored dominant dpy-10 gain-of-function mutation. Heterozygous F1dpy-10(gof) animals are roller (Rol) and homozygous dpy-10(gof) animals are dumpy (Dpy). The presence of the phenotype indicates that Cas9 editing occurred in these animals and improves the odds of identifying an editing event at the second locus in the Rol or Dpy F1 animals. A successful editing experiment should result in 33-50% of injected P0 worms yielding 20 or more F1 offspring that are Rol or Dpy90. It is then possible to choose non-Rol animals to return dpy-10 to wildtype and select for the homozygous edit of interest. As a rule of thumb, the concentration of the crRNA targeting the co-CRISPR reference gene should be half that of the crRNA targeting the gene of interest. If an edit in the gene of interest is not recovered, the ratios of the two CRISPR RNAs can be adjusted to increase the likelihood of recovering the desired mutation. For instance, increasing the amount of crRNA for the gene of interest relative to the reference gene crRNA will increase the percentage of worms possessing edits in the gene of interest within the population of worms that also possess edits at the reference gene locus. Co-conversion frequencies vary, but the rates are typically 20-60%, often yielding homozygous edits in the F1 generation (Figure 1C).
P. hawaiensis hatchlings that have been edited to knock out the Abdominal-B gene (Abd-B) display clear morphological abnormalities3 (Figure 1D). This gene is required for correct abdominal patterning, and its disruption results in thoracic-type jumping and walking legs replacing the swimming and anchor legs that are usually present on the abdomen.
Determining genome editing outcomes at the genotypic level requires either sequencing or an in vitro assay that detects sequence changes. Here, we show representative sequencing data from our model cell types and organisms, highlighting different approaches to editing quantification. Note that the figure labels are generalized because all methods shown here can be applied to any biological system.
Sequencing-based approaches vary in technical complexity and depth of results. For clonal edited populations or easily-separable individual organisms, edited individuals can be sequenced following genomic DNA extraction. Standard Sanger sequencing results will reveal the sequence change at the Cas9-cut site in a given individual, with hypothetical frameshifts that would disrupt its function (Figure 2A). The online tool used for sequencing is another Sanger sequencing-based approach that can be applied to mixed populations rather than individual mutants78. Sequences are analyzed with an online tool that can approximate overall editing efficiency as well as predominant sequence outcomes. The representative data are shown in Figure 2B.
The most thorough sequencing method described here is deep sequencing (sometimes referred to as high-throughput or next-generation sequencing). This method provides DNA sequences from individual genomes in a mixed population. Such data can be illustrated in a variety of ways. Here, we have classified individual sequencing reads from edited cells based on the editing outcome (Figure 2C). Most cells are edited via the NHEJ pathway, which typically results in gene disruption. In others, the target gene has been swapped out for an alternate version via HDR27.
<img alt="Table 1" src="/files/ftp_upload/57350/57350tbl1v2.jpg" /> 
Table 1: Positive controls for preliminary genome editing experiments. This table shows the key information needed to perform a first-time genome editing experiment in each of the cells and organisms described in this protocol. Following these parameters is likely to yield a successful result that can be used to test the protocol or as a baseline for comparison once the experimenter is targeting a gene of their own interest. F: forward, R: reverse, HDR: homology-directed repair.&#160;<a href="/files/ftp_upload/57350/Farboud_et_al_Table_1.xlsx" target="_blank">Please click here to download this table.</a>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57350/57350fig1v2.jpg" /> 
Figure 1: Representative phenotypic results from Cas9 RNP editing of primary human cells and organisms. (A) This is an HPLC trace showing that after successful genome editing, HSPCs that are differentiated into late-stage erythroblasts will produce more functional hemoglobin than sickle hemoglobin. Mutant erythrocytes produce sickle hemoglobin (HbS), while successfully-edited cells will produce healthy hemoglobin (HbA and HbA2) as well as fetal hemoglobin (HbF). The absorbance is graphed in arbitrary units (au). This panel was first published in DeWitt et al.27. It is reprinted with permission from the American Association for the Advancement of Science. (B) On the left, for each condition, this panel shows flow cytometry data showing that surface-stained T cells do not express CD25 after the CD25 gene has been knocked out with RNP. The CD25 abundance is plotted on the x-axis with the cell size on the y-axis. On the right, for each condition, this panel shows the Phospho-Stat5 (pStat5) quantification after an induction with IL-2. The signaling is reduced when the IL-2 receptor is absent (CD25 KO). The pStat5 abundance is plotted on the x-axis and the data resulting from three different levels of IL-2 input are compared vertically. (C) This panel shows a Caenorhabditis elegans co-CRISPR/co-conversion screen targeting dpy-10 as the co-conversion marker. Two guide RNAs target two loci, dpy-10 and your favorite gene (yfg), in the same P0-injected animal. HDR at dpy-10 results a Rol or Dpy phenotype. The selection of Rol- or Dpy-F1 animals increases the chances of identifying edits at the second locus. (D) This panel shows that wildtype Parhyale hawaiensis hatchlings have normal abdomens with swimming and anchor legs. The Abd-B knock-out hatchlings (F0 individuals) develop an abdomen transformed towards thorax. Thus, the swimming and anchors legs are gone and replaced by the jumping and walking legs associated with a normal thorax. <a href="/files/ftp_upload/57350/57350fig1large.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/57350/57350fig2v2.jpg" /> 
Figure 2: Typical results from editing outcome analysis methods. (A) This panel shows examples of the Sanger sequencing results from individual F1&#160;P. hawaiensis organisms, including the wildtype sequence and three different indels that disrupt the gene function by shifting the open reading frame. (B) These TIDE results show the range of insertions and deletion events that occurred at a Cas9-target site in a pool of sequenced T cells. The x-axis indicates the length of a given insertion or deletion in nucleotides. (C) These deep sequencing results show no genome editing without nucleofection or gRNA, and successful editing with intact Cas9 RNP, grouped by DNA repair outcome in HSPCs. <a href="/files/ftp_upload/57350/57350fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms at JoVE.com

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

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 ...

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33.6K Views.  University of California, Berkeley. Met behulp van een voorgemonteerd Cas9 is ribonucleoprotein complex (RNP) een krachtige methode voor het bewerken van nauwkeurige en efficiënte genoom. Hier, wijzen wij op het nut ervan over een breed scala van cellen en organismen, met inbegrip van de primaire menselijke cellen en beide klassieke en opkomende modelorganismen.

Video: Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

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 ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

Cancer Research

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

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 ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

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 ...

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4&#945; locus.

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus in human embryonic stem cells (hESCs).

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing ...

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species alike. This technology can be used for a variety of genome editing approaches, from gene knockouts and knockins to more specific changes like the introduction of a few nucleotides at a targeted location. Genome editing can be used for a multitude of applications, including the partial functional characterization of genes, the production of transgenic organisms and the development of diagnostic tools. Compared to previously available gene editing strategies, the CRISPR-Cas9 system has been shown to be easy to establish in new species and boasts high efficiency and specificity. The primary reason for this is that the editing tool uses an RNA molecule to target the gene or sequence of interest, making target molecule design straightforward, given that standard base pairing rules can be exploited. Similar to other genome editing systems, CRISPR-Cas9-based methods also require efficient and effective transformation protocols as well as access to good quality sequence data for the design of the targeting RNA and DNA molecules. Since the introduction of this system in 2013, it has been used to genetically engineer a variety of model species, including Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Mus musculus. Subsequently, researchers working on non-model species have taken advantage of the system and used it for the study of genes involved in processes as diverse as secondary metabolism in fungi, nematode growth and disease resistance in plants, among many others. This protocol detailed below describes the use of the CRISPR-Cas9 genome editing protocol for the truncation of a gene involved in the sexual cycle of Huntiella omanensis, a filamentous ascomycete fungus belonging to the Ceratocystidaceae family.

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is an easy-to-use genome editor that has been used in model and non-model species. Here we present a protein-based version of this system that was used to introduce a premature stop codon into a mating gene of a non-model filamentous ascomycete fungus.

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

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