Bioinformatics Mastery: Genome Engineering using CRISPR Cas9

Explore genome editing with engineered nucleases, from zinc finger nucleases and TALENs to CRISPR-Cas9, including how DNA cuts and cellular repair enable precise genetic changes.

This lecture explains non-homologous end joining, the dominant double-strand break repair pathway, detailing end processing that yields insertions and deletions and contrasts with microhomology-based repair and homologous directed repair.

Learn the homology-directed repair pathway for precise genome editing, including 5' to 3' resection, strand invasion, and displacement loop formation, with donor DNA templates and cell-cycle constraints enabling error-free repair.

Learn how restriction enzymes cut DNA at recognition sequences, defend host DNA by methylation in the restriction-modification system, and how type II enzymes separate cleavage and recognition domains for cutting.

Explore zinc finger nucleases, engineered dna binding and cleavage domains that create targeted double-stranded breaks for genome editing, repaired by non-homologous end joining or homologous recombination.

Explore TALENs, transcription activator like effector nucleases, using 33-34 amino acid repeats with repeat variable diversity (RBD) and FokI cleavage for targeted genome editing.

Explore how crispr cas9 enables genome editing, guided by pam and a guide rna, to cleave targeted dna. Trace its origins in bacterial immunity, from crispr repeats to cas9.

Explore how CRISPR Cas9 enables genome engineering in eukaryotic cells by using Cas9, guide RNA, and PAM to induce targeted double-strand breaks, guiding HDR or NHEJ repair.

Learn to retrieve the mRNA sequence from the p53 gene by navigating the NCBA site, accessing reference sequences, downloading the fasta format, and saving the nucleotide data.

Design CRISPR Cas9 guide RNAs using CRISPR Direct, inputting a nucleotide sequence and setting PAM for specificity checks. Assess highly specific targets and review off-target notes and seed regions.

Retrieve exon sequences from a gene by selecting coding regions, then download the exon sequence for sdr design.

Learn to design CRISPR Cas9 guide RNAs with the WU-CRISPR tool, inputting gene symbols or target sequences, and evaluating potency and off-target predictions.

Learn to design CRISPR-Cas9 guide RNA with the Cas-designer tool, input sequences, use the default PAM, and interpret target positions, GC content, mismatches, and out-of-frame scores to select targets.

Develop proficiency in predicting CRISPR-Cas9 guide RNA with the scan tool, cross-check results across multiple tools, and interpret target and off-target rankings to select the best guide sequence.

Explore off-target analysis with Off-Spotter for CRISPR-Cas9 guide RNA design, input sequences, evaluate mismatches and 20-mer targets, and compare results to select the best zero-mismatch options.

Design CRISPR Cas9 guide RNAs for human p53 using guides, set library size, view exons, export a CSV, and evaluate CFD, GC, on-target efficiency, and off-targets to select the guide.

Explore a sgRNA designer tool for CRISPR-Cas9 genome editing, learning to search by gene or sequence, evaluate intrinsic and specificity scores, and select the most efficient guide.

Design CRISPR Cas9 gRNA using the Jilani design checker, preparing FASTA sequences for Homo sapiens and evaluating on-target and off-target scores to pick the best gRNA.

Design CRISPR-Cas9 guides with Chop Chop, evaluate mismatches and efficiency, view rankings and primers, and select the best knockout target with zero mismatches.

Use the CRISPR tool to design CRISPR Cas9 guide RNAs, input sequences, select a genome, and evaluate specificity, PCR primers, and predicted efficiency and off-targets.