UcanCRISPR online: CRISPR/Cas9 master class

Explore the discovery and repurposing of CRISPR as a bacterial adaptive immune system. Learn how CRISPR identifies targets and defends against pathogens, and explore the evolution of six CRISPR types.

Explore core vocabulary for DNA interactions, ribonucleoprotein complexes, and CRISPR components, including crRNA, spacer targets, Cas proteins, RNA guides, tracrRNA, and sgRNA, plus the acquisition, processing, and interference steps.

Explore four key mental models of CRISPR processes, including the adaptive immunity steps, RNA processing, Cas9 activation and DNA targeting, and spacer acquisition via Cas1/Cas2.

Explore how Cas9 acts as an RNA-guided DNA nuclease, using guide RNA and tracrRNA derived from crRNA to recognize and cut foreign DNA.

Explore how CRISPR-Cas9 adapts to target pathogens, guided by PAM motifs and spacer-protospacer matching, and how interference cleaves viral DNA in bacterial immunity.

Explore how CRISPR was discovered in nature and repurposed for human utility, detailing gRNA design, U6 promoter initiation, sgRNA processing, and endo mutations.

Explore how Cas1 and Cas2 drive spacer acquisition in the Cascade CRISPR system, including palindrome repeats and insertion mechanisms as a cellular recording device.

Examine crRNA processing in CRISPR-Cas adaptive immunity. Learn how Csy4-like nucleases cleave pre-crRNA after hairpin structures to form interference complexes and release individual crRNAs.

Explore modular Cas proteins and their multifunctionality in CRISPR systems, including interference proteins that process RNA. Examine Cas1, Cas6, and reverse transcriptase–Cas6 fusion enabling RNA or DNA-based spacer acquisition.

Explore the evolution and classification of CRISPR systems, from class 1 and class 2 architectures to types 1-6, and how viral interactions drive spacer acquisition and diversity.

Trace the proposed origin of CRISPR systems, where virus-fighting and innate immunity genes form a CRISPR array with Cas proteins, building class one and later class two systems.

Describe how class 1 crispr systems are multi-protein complexes, mainly types 1 and 3 in bacteria, and show structural similarity that relates to dna target binding and dna cutting.

Explore class 2 CRISPR systems, including type 2, 5, and 6, with single-protein effectors that target DNA and RNA, offering a powerful toolbox for genetic engineering.

Explore the diversity and universality of CRISPR RNA across systems, including different repeat sequences and structures, tracrRNA requirements, RNA processing mechanisms, and how target cuts vary relative to the pan.

Explore how CRISPR/Cas9 creates targeted DNA breaks and how cells repair them via non homologous end joining or homologous directed repair, using repair templates for precise genome edits.

Explore nickases and split Cas9 strategies to steer CRISPR-Cas9 edits, including single-strand cuts, offset target sites, and regulated dual-half systems activated by rapamycin.

Identify CRISPR-induced mutations through screening and sequencing, using genotype checks and reporter gene assays. Employ phenotypic selection and resistance markers to enrich and confirm edits.

Learn how to detect CRISPR-induced mutations using endonuclease screens, PCR amplification, and heteroduplex analysis with surveyor nuclease and T7 endonuclease I assays, plus restriction-site loss screening, visualized on a gel.

Detect CRISPR mutations through high resolution melting analysis of PCR products, observing melting temperature shifts from deletions or insertions, followed by Sanger sequencing for single-sample confirmation.

Assess CRISPR off-targets by analyzing seed region near PAMs and applying unbiased high-throughput screening methods such as bluffs, guide seek, and did you genome sequence seek to map DNA breaks.

Explore how CRISPR-Cas9 serves as a versatile platform for genetic engineering, repurposing cutting and non-cutting Cas9 to repress or activate transcription, guide RNA, and target DNA or RNA sequences.

Demonstrate how dead dCas9 mutants (D10A, H840A) block transcription and recruit chromatin remodeling to repress gene expression, enabling CRISPR interference and transcriptional modulation.

Activate transcription with CRISPR-Cas9 by recruiting RNA polymerase through activation domains such as VP64 and p300, and assemble multi-component transcriptional activation complexes with the MS2 RNA motif across diverse hosts.

CRISPR/dCas9 targets gene promoters to map DNA–protein interactions using ChIP, ChIP-seq, ChIP-MS, and cast-back labeling to identify nearby proteins by mass spectrometry.

Explore Cas9 structure and its two-lobed biloba form, guiding improved enzymes and variants with relaxed PAM requirements. RNA loading and DNA binding trigger conformational changes that activate the cutting site.

Cas9 undergoes conformational changes upon sgRNA and DNA binding, tests the PAM and seed region, and promotes an hour loop via RNA-DNA pairing.

Explore Cas9 structure and function, engineered variants for higher accuracy, and the stepwise RNA–DNA interactions that enable selective cleavage and proofreading to prevent off-target effects.

Explore strategies to engineer Cas9 proteins with relaxed and expanded PAM requirements, featuring SC cast nine variants, directed evolution, and phage-assisted selection to boost accuracy and target range.

Learn base editing with Cas9 fusions to make precise single-base changes, enhance homologous recombination for custom sequences, and store information in crispr by iterative spacer addition using genetic circuits.

Cas9 fusion with HDR promoting proteins enhances site-specific recombination and homologous recombination repair, enabling targeted DNA exchange and genome rewriting with repair templates.

Discover CRISPR/Cas9 genetic recording, where spacer acquisition encodes information in bacterial genomes, enabling image and video data storage through protospacer regions, PAM-driven updates, and high-throughput retrieval.

Explore engineered genetic circuits that use transcription and translation to form a two-input logic gate, where promoters drive Cas9 and sgRNA expression in bladder cancer cells, with a luciferase readout.

Explore ethical considerations of CRISPR, including safety, informed consent, justice and equity, and embryo gene editing, with a personal responsibility paradigm for research.

Focus on safety through governance, containment, surveillance, and reversible options to mitigate crispr-related risks; emphasize anti-crispr countermeasures and robust, affordable monitoring, with open-source non-ip-restricted access to prevent gatekeeper control.

Explore gene drives as a mechanism that copies itself between chromosomes, converting heterozygotes to homozygotes in Anopheles mosquitoes. Examine safety, containment, and concerns, noting CRISPR infancy and keeping deployment hypothetical.

Harness CRISPR/Cas9 to modify somatic cells through ex vivo and in vivo approaches. Examine the ethical, consent, and regulatory boundaries around germline edits and the potential for heritable changes.

Identify potential cas interference proteins from genomic data and develop an open source crispr released into the public domain under an mda, focusing on type 2 and 5 single-protein designs.

Explore the crispr design process, from identifying a target site and designing sgRNA to introducing the genetic construct and screening transformants for the desired mutation.

Explore CRISPR/Cas9 configurations, from plasmid-based expression of Cas9 and gRNA to RNA formats and RNP delivery, with pros, cons, and strategies for genome targeting.

Learn mutation screening in CRISPR experiments with PCR amplification, mismatch nuclease detection, sequencing confirmation, and protein expression checks with a reporter fusion or transcript level checks via RT-qPCR, noting off-targets.

Explore in vitro transcription to produce sgRNA transcripts from a DNA template with a T7 promoter, and learn about commercially available sgRNA, tracer RNA, chemically modified crRNA, and Cas9 compatibility.

Transform CRISPR components through the cell membrane and wall into the nucleus using viral delivery, electroporation, gene guns, chemical methods, lipofection, or microinjection.

Design sgRNA sequences for CRISPR/Cas9 by selecting target sites based on gene goal (knockout, activation, or tagging) and PAM constraints, while minimizing off-targets and GC considerations using automated tools.

Design, build, and test a CRISPR/Cas9 vector for the microalga Nannochloropsis, including plasmid design, gRNA targeting, transformation by electroporation, and selection to obtain non transgenic edited lines.

Design and apply CRISPR with purified Cas9 or Cas12a RNPs in Xenopus to disrupt the tyrosinase gene and pigmentation. Learn guide design, ordering components, microinjection, and PCR screening.