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We thank Beatriz Olalla Sastre for helpful discussions relating to this protocol. This work was supported by the British Heart Foundation&#8217;s Big Beat Challenge award to CureHeart (award reference no. BBC/F/21/220106), the Cystic Fibrosis Foundation (award NEWBY23XX0), and the US National Institutes of Health (R00HL163805 and DP2OD038783 to G.A.N.).&#160;J.N.W. acknowledges support by NIH Grant No. T32 GM148383.

<ol>
	<li>Gao H, Gao S, Kan G, Valentovich LN, An Y. Research progress of base editing and prime editing tools based on the CRISPR/Cas system. Mol Ther Nucleic Acids. 2025;36.</li>
	<li>Chen PJ, Hussmann JA, Yan J, et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell. 2021;184(22):5635&#8211;5652. doi:10.1016/j.cell.2021.09.018.</li>
	<li>Newby GA, Yen JS, Woodard KJ, et al. Base editing of haematopoietic stem cells rescues sickle cell disease in mice. Nature. 2022;595(7866):295-302. doi:10.1038/s41586-021-03609-w.</li>
	<li>Sousa AA, Hemez C, Lei L, et al. Systematic optimization of prime editing for the efficient functional correction of CFTR F508del in human airway epithelial cells. Nat Biomed Eng. 2024;9(1):7. doi:10.1038/s41551-024-01233-3.</li>
	<li>Mayuranathan T, Newby GA, Feng R, et al. Potent and uniform fetal hemoglobin induction via base editing. Nat Genet. 2023;55(7):1210-1217.</li>
	<li>He W, Zhang X, Zou Y, et al. Effective synthesis of high-integrity mRNA using in vitro transcription. Molecules. 2024;29(11).</li>
	<li>Popova PG, Lagace MA, Tang G, Blakney AK. Effect of in vitro transcription conditions on yield of high-quality messenger and self-amplifying RNA. Eur J Pharm Biopharm. 2024;198.</li>
	<li>Zhang F, Wang Y, Wang X, et al. RT-IVT method allows multiplex real-time quantification of in vitro transcriptional mRNA production. Commun Biol. 2023;6(1).</li>
	<li>Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E. CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 2014;42(W1)-W407.</li>
	<li>Concordet JP, Haeussler M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 2018;46(W1)-W245.</li>
	<li>Mohr SE, Hu Y, Ewen-Campen B, Housden BE, Viswanatha R, Perrimon N. CRISPR guide RNA design for research applications. FEBS J. 2016;283(17):3232-3245.</li>
	<li>Huang TP, Newby GA, Liu DR. Precision genome editing using cytosine and adenine base editors in mammalian cells. Nat Protoc. 2021;16(2):1089-1106. doi:10.1038/s41596-020-00450-9.</li>
	<li>Doman JL, Sousa AA, Randolph PB, Chen PJ, Liu DR. Designing and executing prime editing experiments in mammalian cells. Nat Protoc. 2023;18(6):1780-1818. doi:10.1038/s41596-022-00724-4.</li>
	<li>Chicaybam L, Barcelos C, Peixoto B, et al. An efficient electroporation protocol for the genetic modification of mammalian cells. Front Bioeng Biotechnol. 2017;4:99.</li>
	<li>Clement K, Rees H, Canver MC, et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat Biotechnol. 2019;37(3):224-230.</li>
</ol>

N.W. and E.V.B. have filed patent applications on genome editing technologies through Johns Hopkins University. G.A.N. has filed patent applications related to genome editing technologies through the Broad Institute and Johns Hopkins University.
Authors contribution: 
M.V., J.N.W., E.V.B, D.M.E., J.D., and G.A.N. each drafted separate sections of the text. M.V. assembled and edited the final text followed by edits from all authors.

This protocol provides a workflow for genome editing in primary mammalian cells using electroporation of editor mRNA and synthetic guide RNAs. Delivery as mRNA tends to yield more efficient editing relative to plasmid delivery, and mRNA tends to be simpler and more consistent between batches relative to editor protein or ribonucleoprotein. When mRNA and ribonucleoprotein forms of base editors have been compared head-to-head in some prior work, mRNA has yielded better editing efficiency3. Compared to plasmid or viral delivery methods, mRNA-based delivery enables transient expression and reduced risk of genomic integration, although it may require optimization for different cell types. In addition to lower editing efficiency, the use of plasmid DNA for genome editing carries a risk of random integration into the genome of target cells and can increase the risk of off-target editing due to prolonged expression of the editor. The combination of targeted amplicon sequencing and CRISPResso2 enables standardized quantification of editing efficiency and characterization of any allelic outcomes3. &#160;
Several factors are vital for success. Critical steps in this protocol include maintaining RNA integrity through strict RNase-free handling, selecting optimal guide RNAs, and carefully optimizing electroporation conditions for each cell type. RNA integrity and careful RNase control strongly influence outcomes, and guide RNA selection remains a primary determinant of efficiency and byproduct profiles. Electroporation settings require empirical optimization by cell type and some programs that yield high editing may also cause toxicity. If low editing efficiency is observed, optimization of electroporation parameters, RNA quality, or guide RNA design should be considered. If excessive toxicity occurs, reducing electroporation strength or RNA input may improve cell viability. Additional optimization strategies include testing alternative electroporation buffers and programs tailored to the target cell type or modifying editor or guide RNA concentrations. This workflow provides a practical platform to benchmark editors and produce modified cells before downstream single-cell cloning, phenotyping, in vivo studies, or translational development. This workflow is broadly applicable for evaluating genome editing strategies in primary cells, enabling systematic comparison of editing modalities across genomic contexts and cell types. It supports disease modeling through the introduction or correction of clinically relevant variants, as well as functional interrogation of coding and non-coding regulatory elements. In addition, it provides a platform for optimizing therapeutic editing approaches, including assessment of on-target efficiency, off-target activity, and editing durability. The workflow can also be used to screen and prioritize guide RNAs and editing systems, evaluate delivery strategies, and test patient-specific variants in a precision medicine framework. Collectively, it serves as a versatile bridge between basic genome biology, translational research, and preclinical development. A limitation of this method is that electroporation efficiency and cell viability can vary substantially between cell types, requiring empirical optimization. In addition, editing outcomes may depend on guide RNA design and purity, and the accessibility of the target locus, which may limit reproducibility across different experimental systems. Despite these constraints, careful optimization of experimental parameters and guide design can mitigate variability, enabling reliable and reproducible application of this approach across diverse cellular systems.

Efficient genome editing in primary mammalian cells supports the generation of genetic models and the preclinical evaluation of candidate therapeutic strategies. Recent advances in CRISPR-based technologies, including base editing and prime editing, enable precise nucleotide modifications without introducing double-strand DNA breaks, thereby improving the purity of editing outcomes1,2. These technologies have been successfully applied in disease modeling and therapeutic genome editing studies, including strategies to correct variants that lead to sickle cell disease3 and cystic fibrosis4.
A particularly efficient strategy to deliver genome editing components to mammalian cells is electroporation of in vitro transcribed (IVT) mRNA encoding the editing enzyme combined with synthetic guide RNAs (gRNAs). Delivery of mRNA enables transient expression of genome editors, minimizing prolonged nuclease activity and reducing the likelihood of off-target editing events. Advances in IVT technology have improved the efficiency, yield, and quality of mRNA synthesis4,5,6. Plasmid DNA templates and PCR-derived DNA templates are each commonly used for in vitro transcription and each present distinct advantages and limitations. In this protocol, PCR-generated templates are preferred to avoid issues associated with propagating long poly(A) tracts in plasmids, which are prone to recombination and length variability. However, using PCR templates as described in this protocol does require an expensive reverse primer that harbors the 120-nucleotide poly(A) tail, and PCR may be more error prone than bacterial propagation of plasmids for sequences not encoded in the primers. As an alternative, commercial providers such as Elegen and 4basebio can supply high-fidelity DNA templates, offering a reliable, though more costly, option for generating transcription templates.
Careful guide RNA design is another critical component of successful genome editing experiments. Several computational tools have been developed to assist researchers in identifying optimal gRNA sequences based on predicted on-target efficiency and minimal off-target activity. Widely used platforms include CHOPCHOP and CRISPOR, which enable identification of guide sequences compatible with protospacer adjacent motif (PAM) requirements of Cas enzymes and provide scoring metrics to prioritize candidate guides with high predicted activity and specificity7,8. The fundamental principles and best practices for guide RNA design have been extensively described in the CRISPR literature9,10,11,12,13.
Electroporation is commonly used to deliver genome editing reagents to mammalian cells because it can achieve high delivery efficiency in cell types that are typically resistant to lipid-based transfection approaches. Electroporation transiently permeabilizes the cell membrane, allowing exogenous nucleic acids such as mRNA and gRNAs to enter the cytoplasm. Optimized electroporation protocols have been developed for multiple mammalian cell types, including primary cells, fibroblasts, lymphoblastoid cell lines, and induced pluripotent stem cells14. Commercial electroporation platforms such as the Lonza 4D and Amaxa systems provide cell-type-specific programs and reagents designed to improve delivery efficiency while maintaining cell viability. High-throughput electroporation systems such as the Amaxa 96-well Shuttle platform further enable scalable genome editing experiments across multiple experimental conditions. The Neon electroporation system is also commonly used but is not the focus of this protocol.
Following delivery of genome editing components, editing outcomes are typically assessed using targeted next-generation sequencing (NGS) of the genomic region surrounding the editing site. Sequencing libraries are generated by PCR amplification of the target locus, followed by indexing and high-throughput sequencing. The resulting sequencing reads are analyzed using specialized bioinformatic pipelines designed for genome editing experiments. In this protocol, editing outcomes are quantified using CRISPResso2, a widely used computational platform that enables detailed analysis of genome editing outcomes from sequencing data15. CRISPResso2 aligns sequencing reads to a reference sequence and quantifies editing outcomes, including overall editing efficiency, insertion&#8211;deletion (indel) frequencies, precise nucleotide substitutions, and editing distributions across the target region. The software also provides graphical visualizations and statistical summaries that facilitate comparison between experimental conditions and optimization of editing parameters.
Together, the integration of high-quality IVT mRNA production, optimized delivery by electroporation, and high-resolution sequencing analysis provides a robust workflow for evaluating genome editing strategies in cultured cells (Figure 1). This approach enables iterative optimization of editing efficiency, specificity, and experimental parameters prior to advancing genome editing strategies toward in vivo studies and potential therapeutic applications.

<table><tbody><tr><td>1M Tris-HCl, pH 8.0</td><td>Thermo Fisher Scientific</td><td>15568025</td><td></td></tr><tr><td>Agarose&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>R0491&amp;nbsp;</td><td>Agarose UltraPure 16500500 ThermoFisher 500g</td></tr><tr><td>AMPure XP SPRI Beads&amp;nbsp;</td><td>Beckman Coulter&amp;nbsp;</td><td>B23317&amp;nbsp;</td><td>A63880, AMPure XP Beads for DNA Cleanup, 5 mL, Beckman Coulter</td></tr><tr><td>ATP (100 mM)&amp;nbsp;</td><td>New England Biolabs&amp;nbsp;</td><td>N0450&amp;nbsp;</td><td></td></tr><tr><td>CEPT Cocktail&amp;nbsp;</td><td>Bio-Techne / Tocris&amp;nbsp;</td><td>7200&amp;nbsp;</td><td></td></tr><tr><td>CleanCap AG Reagent&amp;nbsp;</td><td>TriLink Biotechnologies&amp;nbsp;</td><td>N-7113&amp;nbsp;</td><td>CleanCap AG 3&amp;prime; OMe CleanScript IVT Kit, 125 x 20 &amp;micro;L rxns,&amp;nbsp;</td></tr><tr><td>CTP (100 mM)&amp;nbsp;</td><td>New England Biolabs&amp;nbsp;</td><td>N0450&amp;nbsp;</td><td></td></tr><tr><td>Custom DNA Primers&amp;nbsp;</td><td>Integrated DNA Technologies&amp;nbsp;</td><td>Custom&amp;nbsp;</td><td></td></tr><tr><td>DNA Loading Dye&amp;nbsp;</td><td>New England Biolabs&amp;nbsp;</td><td>B7024&amp;nbsp;</td><td></td></tr><tr><td>DPBS (Dulbecco&amp;rsquo;s Phosphate Buffered Saline)&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>14190-144&amp;nbsp;</td><td>D8537-100ML</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>11965-092&amp;nbsp;</td><td>DMEM High Glucose [4,500 mg/L] with [4.0 mM] L-Glutamine and [110 mg/L] Sodium Pyruvate, 500 mL</td></tr><tr><td>Gel Extraction Kit&amp;nbsp;</td><td>Qiagen&amp;nbsp;</td><td>28704&amp;nbsp;</td><td></td></tr><tr><td>GTP (100 mM)&amp;nbsp;</td><td>New England Biolabs&amp;nbsp;</td><td>N0450&amp;nbsp;</td><td></td></tr><tr><td>Illumina Indexing Primers&amp;nbsp;</td><td>Illumina&amp;nbsp;</td><td>FC-131-2001&amp;nbsp;</td><td>10uM stocks</td></tr><tr><td>Illumina MiSeq Sequencer&amp;nbsp;</td><td>Illumina&amp;nbsp;</td><td>SY-410-1003&amp;nbsp;</td><td></td></tr><tr><td>Lithium Chloride RNA Precipitation Solution&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>AM9480&amp;nbsp;</td><td></td></tr><tr><td>Lonza 4D Nucleofector System&amp;nbsp;</td><td>Lonza&amp;nbsp;</td><td>AAF-1002X&amp;nbsp;</td><td></td></tr><tr><td>ssRNA Ladder NEB</td><td>NEB</td><td>N0362S</td><td>5ul/ reaction</td></tr><tr><td>MiSeq Reagent Kit v3 (300 cycles)&amp;nbsp;</td><td>Illumina&amp;nbsp;</td><td>MS-102-3002&amp;nbsp;</td><td></td></tr><tr><td>N1-Methyl-Pseudouridine-5&amp;prime;-Triphosphate&amp;nbsp;</td><td>TriLink Biotechnologies&amp;nbsp;</td><td>N-1081&amp;nbsp;</td><td></td></tr><tr><td>NEBNext Ultra II Q5 Master Mix&amp;nbsp;</td><td>New England Biolabs&amp;nbsp;</td><td>M0544&amp;nbsp;</td><td></td></tr><tr><td>Nuclease-Free Water&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>AM9937&amp;nbsp;</td><td></td></tr><tr><td>NucleoCounter NC Slide A8&amp;nbsp;</td><td>ChemoMetec&amp;nbsp;</td><td>941-0008&amp;nbsp;</td><td></td></tr><tr><td>NucleoCounter NC-3000&amp;nbsp;</td><td>ChemoMetec&amp;nbsp;</td><td>NC-3000&amp;nbsp;</td><td>10ul of cell solution and 0.4167 &amp;mu;L of DAPI solution&amp;nbsp;</td></tr><tr><td>Solution 15, Hoechst 33342, 1 mL 910-3015 Chemometec</td><td>Chemometec</td><td>910-3013&amp;nbsp;</td><td>500 &amp;mu;g/ml ~ 0.9 mM ~ 0.05%</td></tr><tr><td>Nucleocuvette Strips&amp;nbsp;</td><td>Lonza&amp;nbsp;</td><td>V4XP-3032&amp;nbsp;</td><td></td></tr><tr><td>P3 Primary Cell Nucleofector Solution&amp;nbsp;</td><td>Lonza&amp;nbsp;</td><td>V4XP-3032&amp;nbsp;</td><td>1 x 0.675 mL P3 Primary Cell Nucleofector&amp;reg; Solution</td></tr><tr><td>Phusion High-Fidelity DNA Polymerase&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>F530L&amp;nbsp;</td><td></td></tr><tr><td>Proteinase K&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>EO0491&amp;nbsp;</td><td>Quantity: 1mL, &gt;600 U/mL (~20 mg/mL)</td></tr><tr><td>Q5 High-Fidelity DNA Polymerase&amp;nbsp;</td><td>New England Biolabs&amp;nbsp;</td><td>M0491&amp;nbsp;</td><td>2,000 units/ml 100 units&amp;nbsp;</td></tr><tr><td>QIAquick PCR Purification Kit&amp;nbsp;</td><td>Qiagen&amp;nbsp;</td><td>28104&amp;nbsp;</td><td></td></tr><tr><td>Qubit dsDNA High Sensitivity Assay Kit&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>Q33230&amp;nbsp;</td><td></td></tr><tr><td>Qubit RNA Broad Range Assay Kit&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>Q10211&amp;nbsp;</td><td></td></tr><tr><td>Qubit&amp;trade; Assay Tubes</td><td>Thermo Fisher Scientific</td><td>Q32856</td><td></td></tr><tr><td>RNA Gel Loading Dye (2x)</td><td>Thermo Fisher Scientific</td><td>R0641</td><td>5ul/ reaction</td></tr><tr><td>RNA Purification Kit&amp;nbsp;</td><td>Zymo Research&amp;nbsp;</td><td>R1017&amp;nbsp;</td><td></td></tr><tr><td>RNase Away&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>1236B62&amp;nbsp;</td><td></td></tr><tr><td>SDS, 10% solution</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>15553027</td><td></td></tr><tr><td>SYBR Gold Nucleic Acid Gel Stain</td><td>Thermo Fisher Scientific</td><td>S11494</td><td></td></tr><tr><td>SYBR Safe DNA Gel Stain&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>S33102&amp;nbsp;</td><td></td></tr><tr><td>Synthetic sgRNA / pegRNA&amp;nbsp;</td><td>Synthego / IDT&amp;nbsp;</td><td>Custom&amp;nbsp;</td><td>2ug&amp;nbsp;</td></tr><tr><td>HiScribe T7 High Yield RNA Synthesis Kit, 250 reactions</td><td>New England Biolabs&amp;nbsp;</td><td>E2040L</td><td></td></tr><tr><td>TrypLE Express Enzyme&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>12604013&amp;nbsp;</td><td>100 mL</td></tr><tr><td>TE buffer (Tris-EDTA)</td><td>Thermo Fisher Scientific</td><td>&amp;nbsp;AM9849</td><td>TE, pH 8.0, RNase-free Thermo</td></tr><tr><td>Trypsin-EDTA (0.25%)&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>25200056&amp;nbsp;</td><td>1X&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum (FBS), Premium, 500 mL</td><td>Thermo Fisher Scientific</td><td>A5670701</td><td></td></tr><tr><td>mTSER PLUS&amp;nbsp;</td><td>STEM cell Technologies</td><td># 100-0276</td><td></td></tr><tr><td>Penicillin/Streptomycin Mixture 10,000 ug/mL, 100 mL, 4-pack</td><td>Quality Biological</td><td>120-095-721&amp;nbsp;</td><td></td></tr></tbody></table>

genome editing in primary mammalian cells via electroporation of editor rna

Genome Editing in Primary Mammalian Cells via Electroporation of Editor RNA

CRISPR editors including nucleases, base editors, and prime editors can efficiently correct disease-causing genetic variants or disrupt target genes. Editing outcomes are commonly evaluated in cultured primary cells, patient-derived cells, or engineered cell lines to study the impact of genetic variation or as a first step before initiating animal studies or clinical translation. Delivery of editors as mRNA together with synthetic guide RNAs into mammalian cells can improve editing efficiency relative to plasmid-based approaches and prevent issues such as DNA integration or off-target editing from sustained expression. This article presents a workflow to prepare genome editor mRNA by in vitro transcription (IVT), including co-transcriptional capping and chemically-modified nucleotides, electroporate editor mRNA and guide RNAs into primary human fibroblasts, induced pluripotent stem cells (iPSCs), or lymphoblastoid cell lines (LCLs), and quantify editing outcomes by targeted amplicon sequencing on an Illumina platform followed by analysis using CRISPResso2. This workflow enables quantitative benchmarking of guide RNAs, electroporation parameters, and editor variants, and supports downstream applications including single-cell cloning, phenotypic assays, preclinical animal studies, and therapeutic development.

Using an efficient editor with an easily accessed test site in, such as the ABE8e base editor with the HEK3 control site, can often result in editing efficiencies above 90% if the delivery system is well-suited for the cells used in the experiment. Lower editing efficiencies may result if there are problems with the editor mRNA, guide RNA, the target site, or the delivery conditions that make any less well-suited to editing. Using Cas9 variants that access more versatile PAM sequences but bind them less tightly or targeting editors to nucleotides that are outside their ideal editing window, can reduce editing efficiency. Successful IVT-generated editor mRNAs typically appear as a single predominant band with minimal smearing or low-molecular-weight degradation products, indicating high transcript integrity suitable for downstream electroporation (Figure 2).
After electroporation of editor mRNA and synthetic guide RNAs, most cells recover within 24–48 h and maintain their typical morphology with suitable electroporation programs (Figure 3–4). Electroporation conditions should be tailored to the specific cell type, as different buffers can yield markedly different outcomes. The online Lonza Knowledge Center makes recommendations for buffer and electroporation codes for common cell types, and Lonza offers separate optimization kits for cell lines and primary cells. Empirical comparison of buffers can help identify optimal conditions for efficient delivery. Unsuitable programs may lead to no delivery at all or can be highly toxic to cells (losing over 50% or preventing continuation of the cell cycle). Representative optimization experiments demonstrated substantial differences in editing outcomes across electroporation conditions, with some programs yielding high editing efficiencies while others produced minimal editing, highlighting the importance of empirical optimization for each cell type (Figure 3–4).
Across iPSCs, fibroblasts, and LCLs, targeted amplicon sequencing followed by CRISPResso2 analysis is used to quantify editing efficiency and byproducts (Figure 4). Adenine base editing of the HEK3 test locus is shown here (Figure 4), which resulted in the intended nucleotide conversions with low indel frequencies in the analyzed samples. CRISPResso2 analysis enables visualization of edited and unedited alleles at the target locus and facilitates quantification of intended nucleotide conversions, indel frequencies, and other editing byproducts, providing a comprehensive assessment of editing outcomes (Figure 5). Editing efficiency is quantified by measuring the frequency of this precise single-nucleotide substitution at the target site. The HEK3 locus is used as a model locus due to its well-characterized editing profile and reproducibility across experimental systems, enabling robust evaluation of base editing performance under different conditions. We recommend testing this guide as a positive control in human cells when optimizing new editing procedures. Base editing is typically enriched for intended nucleotide conversions with low indel frequencies, whereas prime editing yields a mixture of precise edits and low-frequency byproducts3,12. Editing efficiencies varied across conditions, with clear differences observed between cell types and electroporation programs (Figure 3-4,). These variations likely reflect differences in cellular uptake, viability, and DNA repair activity, as well as the influence of electroporation parameters on delivery efficiency. Collectively, these findings highlight the importance of cell type-specific optimization of electroporation settings to achieve consistent and maximal editing outcomes.
<img alt="mRNA in vitro transcription process; plasmid preparation, PCR, transcription, purification diagram." class="xfigimg" src="/files/ftp_upload/71449/71449fig1.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1. Preparation of editor mRNA by in vitro transcription. 
Schematic overview of editor mRNA generation. (Step 1) Preparation of plasmid template containing the editor coding sequence and T7 promoter. (Step 2) PCR amplification to generate a linear DNA template. (Step 3) In vitro transcription using T7 RNA polymerase to synthesize capped mRNA. (Step 4) Purification of mRNA to remove template DNA, enzymes, and free nucleotides. The resulting capped mRNA is suitable for downstream delivery into mammalian cells by electroporation or lipid nanoparticle. <a href="https://www.jove.com/files/ftp_upload/71449/71449fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Gel electrophoresis results; DNA ladder beside ABE8e, CBE6b, PE2max plasmids; molecular weights." class="xfigimg" src="/files/ftp_upload/71449/71449fig2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2. Example image of mRNA gel electrophoresis  
Electrophoresis image of three genome editor mRNAs run on a 1% agarose gel using SYBR gold stain. The ssRNA ladder from NEB is shown at left with size markers indicated with their length. The lack of substantial smearing or small bands indicates these mRNAs are of suitable quality. <a href="https://www.jove.com/files/ftp_upload/71449/71449fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Gene editing efficiency in human iPSCs; bar chart comparing buffer conditions for optimization." class="xfigimg" src="/files/ftp_upload/71449/71449fig3.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3. Optimization of electroporation conditions for genome editing in induced pluripotent stem cells (iPSCs).  
Editing efficiencies obtained under different electroporation conditions in iPSCs are shown. Cells were electroporated using multiple buffers and program combinations, including CB-150 buffer P4, CD-118 buffer P3, DN-100 buffer P3, DC-100 buffer P3, all on the Lonza 4D Nucleofection device. Editing was assessed using the Neon electroporation device with parameters (1600 V, 20 ms, 1 pulse). Editing efficiency was quantified by targeted sequencing analysis of the edited locus. N=1 per sample. <a href="https://www.jove.com/files/ftp_upload/71449/71449fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Editing optimization bar graph for primary fibroblast culture showing two buffer efficiency results." class="xfigimg" src="/files/ftp_upload/71449/71449fig4.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4. Optimization of electroporation conditions for genome editing in fibroblasts. 
Editing efficiency following electroporation using different nucleofection programs. Fibroblasts were electroporated using multiple Lonza Nucleofector programs (CA-137, CM-138, DS-150, EH-100, EN-150, EO-114, and FF-113), and editing outcomes were quantified by targeted sequencing. Results are shown for two replicates (P2, P3). A negative control without editor delivery is included. Comparison of electroporation codes highlights differences in editing efficiency across electroporation settings, enabling identification of optimal parameters for fibroblast genome editing. N=1 per sample. <a href="https://www.jove.com/files/ftp_upload/71449/71449fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="CRISPR gene editing sequence; diagram with sgRNA, editing window, substitutions, insertions, deletions." class="xfigimg" src="/files/ftp_upload/71449/71449fig5.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5. Characterization of genome editing outcomes at the targeted locus.  
Allele frequency table output from CRISPResso2, which displays the relative abundance of edited and unedited alleles surrounding the sgRNA target region as determined by high-throughput sequencing analysis after editing. Red annotations indicate the base editing window and the pre-existing heterozygous polymorphism present in the cell line used. The guide RNA spacer sequence is underlined in gray. “Predicted cleavage position” shown by a dotted line is shifted relative to the typical “PAM minus 3” position for SpCas9 due to the modification of the window center “wc” in our analysis Batch File (example in Table 8). This does not actually shift the true predicted cleavage location, but the output is shown in this way to indicate that indels surrounding the dotted line within a window of 20 nucleotides will be assessed, capturing both indels centered at the true nick site as well as those centered at the deaminated nucleotides. <a href="https://www.jove.com/files/ftp_upload/71449/71449fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1. Editors and sequences used in this study  
Column 1: Genome editors available on AddGene for in vitro transcription. Names and Addgene IDs for plasmids available that are amenable to in vitro transcription as described in this protocol. Column 2: Sequences of guide RNA spacers used in this study. <a href="https://www.jove.com/files/ftp_upload/71449/Table%201%20v2.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2. PCR reagents for amplification of the mRNA plasmid template.  
The list of reagents and reaction components used for PCR amplification of the plasmid template to generate linear DNA for downstream applications, as well as the sequence of the forward and reverse transcription primers. <a href="https://www.jove.com/files/ftp_upload/71449/Table%202_71449.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3. PCR cycling conditions for amplification of the mRNA template.  
This table lists the thermocycling conditions used for PCR amplification of the mRNA template from the plasmid DNA prior to downstream applications. <a href="https://www.jove.com/files/ftp_upload/71449/Table%203_71449.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4. Reagents used for in vitro. transcription (IVT) reactions.  
This table lists the reagents and reaction components used for in vitro transcription to synthesize editor mRNA from the linear DNA template. An example template sequence (ABE8e) is shown with color-coded elements. <a href="https://www.jove.com/files/ftp_upload/71449/Table%204_71449.xlsx" target="_blank">Please click here to download this Table.</a>
Table 5. Troubleshooting guide for key steps in the genome editing workflow.  
This table summarizes common technical issues that may arise during editor mRNA preparation by in vitro transcription (IVT), and MiSeq sequencing analysis. For each step of the workflow, potential problems, possible causes, and recommended solutions are provided to assist in optimizing editing efficiency and ensuring accurate detection of editing outcomes. <a href="https://www.jove.com/files/ftp_upload/71449/Table%205_Revised%2071449.xlsx" target="_blank">Please click here to download this Table.</a>
Table 6. Optimal electroporation programs for different mammalian cell types.  
This table lists the electroporation program codes and buffer conditions used for efficient delivery of editor mRNA and guide RNAs into different mammalian cell types. The listed electroporation parameters were tested to identify conditions that maximize genome editing efficiency while maintaining cell viability. The same program may not work for each cell line or source of primary cells, so testing alternatives from this list is recommended if editing is initially unsuccessful. <a href="https://www.jove.com/files/ftp_upload/71449/Table%206_Revised%2071449.xlsx" target="_blank">Please click here to download this Table.</a>
Table 7. CRISPResso2 analysis parameters for quantification of genome editing outcomes.  
The first tab lists an example of the HTS1 primer design used for amplification of the target locus. The second tab provides an example of the indexing primers used during library preparation. The third tab illustrates an example of the indexing arrangement used to assign unique indices to each sample during sequencing. The fourth tab contains an example input batch file for CRISPResso2 analysis, and the fifth tab provides an additional example of a CRISPResso2 analysis input Batch File. Note that the window center “wc” is adjusted for base editors to facilitate detection of indels that are centered on the deaminated bases in addition to those centered on the nuclease cut or nick site. <a href="https://www.jove.com/files/ftp_upload/71449/Table%207_Revised%2071449.xlsx" target="_blank">Please click here to download this Table.</a>
Table 8. PCR1 (Tab 1) and PCR2 (Tab 2) reagents for MiSeq library preparation. 
This table lists the reagents and reaction components used in Tab 1 (PCR1) to amplify the target genomic loci and in Tab 2 (PCR2) to incorporate sample-specific index sequences and Illumina sequencing adapters, generating sequencing-ready libraries for MiSeq analysis. <a href="https://www.jove.com/files/ftp_upload/71449/Table%208%20v2.xlsx" target="_blank">Please click here to download this Table.</a>
Table 9. PCR1 (Tab 1) and PCR2 (Tab 2) cycling conditions for MiSeq library preparation.
This table lists the thermocycling conditions used in Tab 1 (PCR1) to amplify the target genomic regions and in Tab 2 (PCR2) to incorporate sample-specific index sequences and Illumina sequencing adapters, generating sequencing-ready libraries for MiSeq analysis. <a href="https://www.jove.com/files/ftp_upload/71449/Table%209%20v2.xlsx" target="_blank">Please click here to download this Table.</a>

The delivery of genome editors as RNAs is a particularly potent means of modifying the genomes of primary mammalian cells and cell lines. The goal of this protocol is to deliver genome editors as mRNA into primary mammalian cells and cell lines, followed by targeted Illumina sequencing and analysis to quantify editing outcomes.

CRISPR editors including nucleases, base editors, and prime editors can efficiently correct disease-causing genetic variants or disrupt target genes. ...

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Research

JoVE Journal

Biology

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

Mouse Genome Engineering Using Designer Nucleases

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.

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

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.

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

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

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

This protocol describes techniques for isolating primary mouse hepatocytes from the liver and electroporating CRISPR-Cas9 as ribonucleoproteins and mRNA to disrupt a therapeutic target gene associated with an inherited metabolic disease of the liver. The methods described result in high viability and high levels of gene modification after electroporation.

Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes

Here, we describe a simple technique intended for the efficient generation of genetically modified mice called CRISPR RNP Electroporation of Zygotes (CRISPR-EZ). This method delivers editing reagents by electroporation into embryos at an efficiency approaching 100%. This protocol is effective for point mutations, small genomic insertions, and deletions in mammalian embryos.

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

This protocol describes the procedure for genome editing in mouse bone marrow-derived macrophages using Cas9-sgRNA ribonucleoprotein complexes assembled in vitro and delivered by electroporation.

High-Efficiency Gene Disruption in Primary Bone Marrow-Derived Macrophages Using Electroporated Cas9-sgRNA Complexes

This protocol presents an optimized approach for producing genetically modified rat models. Adeno-associated virus (AAV) is used to deliver a DNA repair template, and electroporation is used to deliver CRISPR-Cas9 reagents to complete the genome editing process in 2-cell embryo.

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus (AAV) and 2-Cell Embryo Electroporation

The protocol describes methods for Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based epigenome editing in human cell lines using plasmid DNA transfection and mRNA nucleofection.

CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery

The efficiency of the Improved Genome Editing via Oviductal Nucleic Acids Delivery (I-GONAD) method is comparable to traditional microinjection, which requires zygote collection from donor females and transfer to pseudo-pregnant females. This protocol demonstrates its effectiveness by introducing CRISPR/Cas9-induced mutations into the ROSA26 locus on chromosome 6.

Improved Genome Editing via Oviductal Nucleic Acids Delivery-based In Vivo Electroporation Technique for Knockout Mice Generation

Johns Hopkins University School of Medicine.  The delivery of genome editors as RNAs is a particularly potent means of modifying the genomes of primary mammalian cells and cell lines. The goal of this protocol is to deliver genome editors as mRNA into primary mammalian cells and cell lines, followed by targeted Illumina sequencing and analysis to quantify editing outcomes.

Genome Editing in Primary Mammalian Cells via Electroporation of Editor RNA

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