This volume presents a list of cutting-edge protocols for the study of CRISPR-Cas defense systems and their applications at the genomic, genetic, biochemical and structural levels. CRISPR: Methods and Protocols guides readers through techniques that have been developed specifically for the analysis of CRISPR-Cas and techniques adapted from standard protocols of DNA, RNA and protein biology. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Ease of customization. Cas9 can be easily retargeted to new DNA sequences by simply purchasing a pair of oligos encoding the 20-nt guide sequence. In contrast, retargeting of TALEN for a new DNA sequence requires the construction of two new TALEN genes. Although a variety of protocols exist for TALEN construction14,17,48,49, it takes substantially more hands-on time to construct a new pair of TALENs.
We thank B. Holmes for help with computational tools. P.D.H. is a James Mills Pierce Fellow and D.A.S. is a National Science Foundation (NSF) pre-doctoral fellow. V.A. is supported by NIH Training Grants T32GM007753 and T32GM008313. This work was supported by an NIH Director's Pioneer Award (1DP1-MH100706); an NIH Transformative R01 grant (1R01-DK097768); the Keck, McKnight, Damon Runyon, Searle Scholars, Vallee, Merkin, Klingenstein and Simons Foundations; Bob Metcalfe; and Jane Pauley. Reagents are available to the academic community through Addgene and associated protocols; support forums and computational tools are available via the Zhang lab website ( -engineering.org/).
Background: Delivery of CRISPR/Cas RNPs to target cells still remains the biggest bottleneck to genome editing. Many efforts are made to develop efficient CRISPR/Cas RNP delivery methods that will not affect viability of target cell dramatically. Popular current methods and protocols of CRISPR/Cas RNP delivery include lipofection and electroporation, transduction by osmocytosis and reversible permeabilization and erythrocyte-based methods.
Methods: In this study we will assess the efficiency and optimize current CRISPR/Cas RNP delivery protocols to target cells. We will conduct our work using molecular cloning, protein expression and purification, cell culture, flow cytometry (immunocytochemistry) and cellular imaging techniques.
Discussion: This will be the first extensive comparative study of popular current methods and protocols of CRISPR/Cas RNP delivery to human cell lines and primary cells. All protocols will be optimized and characterized using the following criteria i) protein delivery and genome editing efficacy; ii) viability of target cells after delivery (post-transduction recovery); iii) scalability of delivery process; iv) cost-effectiveness of the delivery process and v) intellectual property rights. Some methods will be considered 'research-use only', others will be recommended for scaling and application in the development of cell-based therapies.
In the present chapter, we present the protocols and guidelines to facilitate implementation of CRISPR-Cas9 technology in fungi where few or no genetic tools are in place. Hence, we firstly explain how to identify dominant markers for genetic transformation. Secondly, we provide a guide for construction of Cas9/sgRNA episomal expression vectors. Thirdly, we present how to mutagenize reporter genes to explore the efficiency of CRISPR-Cas9 in the relevant fungus and to ease subsequent CRISPR-mediated genetic engineering. Lastly, we describe how to make CRISPR-mediated marker-dependent and marker-free gene targeting.
After the study completion we will possess the data regarding i) protein delivery and genome editing efficacy; ii) post-transduction recovery; iii) scalability; iv) cost-effectiveness and v) intellectual property burden on the CRISPR/Cas RNP delivery methods to target cells. The methods used will include commercially available transfection reagents, electroporation, transduction by osmocytosis and reversible permeabilization. Also, we will provide optimized protocols for CRISPR/Cas RNP delivery to different cell types (adherent and suspension cells, primary human cells) with enhanced genome editing efficacy.
CRISPR/Cas RNPs will be delivered to target cells using either commercially available transfection reagents, electroporation, transduction by osmocytosis, reversible permeabilization or erythrocyte-based methods. For each delivery technique the optimal ratio (1:1, 1:2 or 1:3) of gRNA to CRISPR/Cas protein required for precomplexing will be determined. Afterwards, the optimal amount of recombinant CRISPR/Cas will be determined. When optimal conditions will be selected, each RNP delivery protocol will be optimized to result in maximal target gene knockout (KO).
There is a little data on CRISPR/Cas RNP delivery to different cell types via chemical membrane permeabilization (for example, iTOP, GSM, reversible permeabilization, etc.). It seems to be scalable, but cell toxicity needs further studies. All reagents used in chemical membrane permeabilization need to be GMP approved to be used in clinical practice and drug development. These methods are very promising as they are relatively cheap and do not require special equipment.
Since then, another generation of high fidelity enzymes have been engineered using cell-based selection approaches. Yeast-based screening systems resulted in evolved Cas9 (evoCas9), which has four mutations in the REC3 domain. evoCas9 has less off-target activity than SpCas9-HF1 or eSpCas9(1.1). Phage-assisted continuous evolution (PACE) methods resulted in xCas9 3.7 which has 7 mutations found in the REC2, REC3, and PAM interacting domains and allows for expanded PAM recognition as well as increased specificity and lower off-target activity. By screening E. coli cells transformed with a pooled library of SpCas9 variants, researchers identified Sniper-Cas, which has less off-target activity than wild type Cas9 and is compatible with truncated gRNAs for increased specificity.
After purification of the locus, molecules associated with the locus can be identified by mass spectrometry (proteins), RNA-sequencing (RNAs), and NGS (other genomic regions). Compared to conventional methods for genomic purification, CRISPR-based purification methods are more straightforward and enable direct identification of molecules associated with a genomic region of interest in vivo.
Once you have selected your target sequences it is time to design your gRNA oligos and clone these oligos into your desired vector. In many cases, targeting oligos are synthesized, annealed, and inserted into plasmids containing the gRNA scaffold using standard restriction-ligation cloning. However, the exact cloning strategy will depend on the gRNA vector you have chosen, so it is best to review the protocol associated with the specific plasmid in question (see CRISPR protocols from Addgene depositors).
STAR (Structured Transparent Accessible Reproducible) Protocols is an open-access peer-reviewed protocol journal from Cell Press that publishes step-by-step experimental and computational protocols from all areas of life, health, earth, and physical sciences.
Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only 25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient.
Between the Neon electroporation system and the Invitrogen Lipofectamine family of reagents, Thermo Fisher Scientific offers complete solutions for all CRISPR transfection needs. With each product, optimized protocols have been developed for ease of delivery and high transfection efficiency. Explore the guide below to find the best fit for your gene editing workflow needs.
In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation."
We published a detailed protocol describing two methods and comparing among three methods for CRISPR knock-in in Drosophila. We also have expertise in knockout and have deposited both knock-in and knock-out cell lines at the Drosophila Genomics Resource Center. Moreover, we have developed CRISPR knockout screening protocols. These are listed below. Please also feel free to contact the Director for more information or consultation.
Our ability to modify the Drosophila genome has recently been revolutionized by the development of the CRISPR system. The simplicity and high efficiency of this system allows its widespread use for many different applications, greatly increasing the range of genome modification experiments that can be performed. Here, we first discuss some general design principles for genome engineering experiments in Drosophila and then present detailed protocols for the production of CRISPR reagents and screening strategies to detect successful genome modification events in both tissue culture cells and animals.
However, one missing information from published data was the robustness of the CRISPR/Cas protocols. In other words, how confident could one be that any desired mutation can be consistently achieved at high frequency? This information becomes particularly relevant when CRISPR/Cas genome editing is applied in high-throughput formats where the screening of several colonies and the repetition of mutagenesis experiments to rescue missing mutants could be impractical. 041b061a72