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Virtual Event:
“CRISPR in Drug Discovery” available for on-demand view

Hear from genome editing experts at our free virtual event which took place on 26 September 2023. Through a selection of interactive speaker sessions and a concluding panel discussion, you’ll discover best practices, tips and tricks for genome editing, the impact of CRISPR in drug discovery, and key considerations for CRISPR screening set-up. 

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CRISPR / Cas9 system

CRISPRs (Clustered Regularly Interspaced Palindromic Repeats) were first discovered in bacteria in the 80s1 and the use of CRISPR-Cas9 gained traction when the potential for side-directed gene knockout was recognized and proposed by Marraffini and Sontheimer in Science 20082. The principle of CRISPR-Cas9 is fairly simple: Cas9 (Cas stands for CRISPR associated) is a dual RNA-guided DNA endonuclease with the ability to generate site-specific DNA doublestrand (ds) breaks. In 2012, Doudna and Charpentier3 – both awarded the Nobel Prize in Chemistry in 2020 – achieved a major breakthrough for this methodology and its implications for genome editing. They combined the before-used precursor CRISPR RNAs (pre-crRNAs) and trans-activating CRISPR RNA (tracrRNA) into a single guide-RNA (sgRNA). With this simplification, it was then possible to have a two component-system consisting of only CRISPR-Cas9 and sgRNA that could introduce double-strand breaks in virtually any gene of the genome. An even easier approach is used when pre-complexing Cas9 with the sgRNA resulting in a ribonucleoprotein (RNP) complex as a single component for site-directed gene modification. 
The ability of CRISPR to introduce specific modifications into the genome allows tremendous flexibility in the choices of targets and assays and allows a precise determination of the drug target as part of functional genomics. In general, CRISPR-Cas9 allows for DNA modifications such as:

  • DNA deletion
  • DNA insertion
  • DNA modification
  • Gene expression activation (CRISPRa)
  • Gene expression repression (CRISPRi)

CRISPR genome editing of resting human CD4+ T cells

White paper and protocol

Ribonucleoprotein (RNP) transfection

Today, genome editing  is a state-of-the-art technology that requires transfection. While plasmids and/or RNAs can be used to achieve genome editing, e.g. to deliver the Cas9 nuclease and the gRNA for CRISPR-based genome editing, also the transfection of ribonucleoproteins (RNPs) is widely used (for CRISPR-based genome editing this requires the transfection of a Cas9-gRNA complex). For further information on RNP transfection download our WhitePaper on Genome Editing of Resting CD4+ T cells or request a copy of the detailed CRISPR transfection protocol.

CRISPR-based genome editing requires co-transfection

For CRISPR transfection, various scenarios are possible: 

  • Transfer of one plasmid carrying both the gRNA and Cas9 nuclease
  • Co-transfection of two separate plasmids (one for the gRNA and one for Cas9)
  • Co-transfection of a plasmid carrying Cas9 and a PCR cassette expressing the gRNA 
  • A Cas9-gRNA ribonucleoprotein (RNP) complex
  • When aiming for insertion or replacement, for all scenarios an additional donor template (e.g. plasmid, single stranded or double stranded oligonucleotide) has to be co-transfected 

Case Study by Roth et al.: Insertion of large DNA sequences in primary human T cells

Featured publications by Roth et al.

Our Nucleofector® Technology has been shown to work as a reliable transfection method for CRISPR-based genome editing tools.

Roth TL et al. (2018) Reprogramming human T cell function and specificity with non-viral genome targeting. Nature, 559(7714):405-409

Roth TL et al. (2020) Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies. Cell, 181(3):728-744.e21

Theo Roth, MD PhD was a speaker at Lonza’s Virtual Event 2022 – Genome Editing of Immune Cells.

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  • High transfection efficiencies for a broad range of cell types, including T cells and iPSCs
  • Efficient co-transfection of various substrates
  • Same conditions for transfecting plasmids, DNA, mRNA, RNPs, PCR cassettes or ss/dsODN

Find further peer-reviewed publications using Nucleofector® Technology for Genome Editing approaches including CRISPR in our Genome Editing Citation List.

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Selected references for genome editing of specific cell types

T cells (human, resting):  Albanese et al. (2021) Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4+ T cells allows complex functional analyses Nat Methods, 19(1):81-89

T cells (human, murine): Seki and Rutz (2018) Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells J Exp Med, 215(3):985-997

HSPCs: Kuppers et al. (2023) Gene knock-outs in human CD34+ hematopoietic stem and progenitor cells and in the human immune system of mice PLoS One, 18(6):e0287052

NK cells (human): Kararoudi et al. (2022) Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV Cell Rep Methods, 18(6):e0287052

Muscle stem cells: Stadelmann et al. (2022) mRNA-mediated delivery of gene editing tools to human primary muscle stem cells Mol Ther Nucleic Acids, 28:47-57

iPSCs: Tanudjojo et al. (2021) Phenotypic manifestation of α-synuclein strains derived from Parkinson's disease and multiple system atrophy in human dopaminergic neurons Nat Commun, 19(1):81-89

Monocytes, macrophages, DCs (human, murine): Freund et al. (2020) Efficient gene knockout in primary human and murine myeloid cells by non-viral delivery of CRISPR-Cas9 J Exp Med, 217(7):e20191692

Various cell lines, e.g. HEK293FT: Ran et al. (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity Cell, 154(6):1380-9

HEK293, HUES62: Ran et al. (2013) Genome engineering using the CRISPR-Cas9 system Nat Protoc, 8(11):2281-2308

Base-editing and prime-editing

As an addition to the CRISPR genome editing toolkit, base and prime editors offer a remarkable potential to correct disease-causing mutations in the human genome1. Both methods do not rely on inducing double-strand breaks (DSBs), thus overcoming associated challenges such as mixtures of insertions and deletions at target sites. Also, the ability to make targeted changes in living cells without relying on homology-directed repair stimulated by DSBs and requiring exogenous donor DNA repair templates, motivates the further exploration of alternative precision genome editing strategies. 

DNA base-editors comprise a catalytically impaired Cas nuclease and a base-modification enzyme that operates on single-stranded DNA. There are two classes of DNA base-editors: cytosine base-editors (CBEs) convert a C•G base pair into a T•A base pair, and adenine base editors (ABEs) convert an A•T base pair to a G•C base pair. Thus, CBEs and ABEs can mediate all four possible transition mutations.

A major limitation of the current base-editing approaches has been the ability to generate precise edits beyond the four transition mutations1. In 2019, the so-called prime-editing was described by Anzalone et al., enabling precise targeted insertions, deletions and all 12 possible classes of point mutations without requiring DSBs or donor DNA templates. The authors conclude that this approach “has the potential to advance the study and correction of the vast majority of pathogenic alleles.”

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Selected publications

Chen L, Park JE, Paa P, Rajakumar PD, Prekop HT, Chew YT, Manivannan SN, Chew WL. Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins. Nat Commun 2021. 12(1):1384

Siegner SM, Ugalde L, Clemens A, Garcia-Garcia L, Bueren JA, Rio P, Karasu ME, Corn JE. Adenine base editing efficiently restores the function of Fanconi anemia hematopoietic stem and progenitor cells. Nat Commun 2022. 12;13(1):6900

Kluesner MG, Lahr WS, Lonetree CL, Smeester BA, Qiu X, Slipek NJ, Claudio Vázquez PN, Pitzen SP, Pomeroy EJ, Vignes MJ, Lee SC, Bingea SP, Andrew AA, Webber BR, Moriarity BS. CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary and immortalized cells. Nat Commun 2021. 12(1):2437

Goodwin M, Lee E, Lakshmanan U, Shipp S, Froessl L, Barzaghi F, Passerini L, Narula M, Sheikali A, Lee CM, Bao G, Bauer CS, Miller HK, Garcia-Lloret M, Butte MJ, Bertaina A, Shah A, Pavel-Dinu M, Hendel A, Porteus M, Roncarolo MG, Bacchetta R. CRISPR-based gene editing enables FOXP3 gene repair in IPEX patient cells. Sci Adv 2020. 6(19):eaaz0571


Anzalone VA, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A and Liu DR. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 2019. 576(7785): 149–157

Gaj T, Gersbach CA, and Barbas CF 3rd ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering Trends Biotechnol 2013. 31(7):397-405

Ishino Y, Shinagawa H, Makino K, Amemura M,and Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product J. Bacteriol. 1987 Dec; 169(12):5429-5433

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, and Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity Science 2012. 337:816-821

Kantor A, McClements ME, and MacLaren RE. CRISPR-Cas9 DNA Base-Editing and Prime-Editing, Int J Mol Sci 2020. 21(17):6240

Marraffini LA and Sontheimer EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA Science 2008. 322:1843-1845

Rees HA and Liu DR. Base editing: precision chemistry on the genome and transcriptome of living cells, Nat Rev Genet 2018. 19(12): 770–788


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