CRISPR Cas System: Applications in Gene Editing and Beyond

Explore how gene editing seeks to cure genetic diseases by repairing mutations in DNA, exemplified by sickle cell disease and the HBB gene, using CRISPR-Cas, TALENs, and zinc finger nucleases.

Learn how non homologous end joining repairs double-stranded breaks by ligating ends after FokI cleavage, often creating insertions or deletions that knock out CCR5 to block HIV entry.

Explore how HDR uses a template DNA with 1 kb homology arms to repair double-stranded breaks and replace IL2Rγ exon 5 with wild-type DNA via zinc finger nucleases and FokI.

Use TALENs, TALE-based DNA-binding repeats fused to a dimeric FokI nuclease, to create targeted 17-base-pair DNA breaks for gene knockout or gene addition.

Compare zinc finger nucleases and TALENs, highlighting TALENs’ greater specificity and lower off-target mutations, yet larger size and delivery challenges; note crispr-cas as the next technology.

Explore crispr cas defense system in bacteria and archaea, creating a memory of prior phage infections by capturing spacers, enabling rapid defense against previously encountered phages.

Explore the CRISPR loci structure, including repetitive palindromic sequences and spacer DNA, and Cas genes and proteins that unwind and cut DNA, forming the system backbone.

Explore the six major CRISPR-Cas types, including Type I’s Cascade and Cas3 DNA degradation, Type II’s tracrRNA-Cas9 system, and Type III’s Csm and Cmr with transcription-dependent targeting.

Explore type IV, V, and VI CRISPR–Cas systems, including the type IV plasmid complex with Csf proteins, Cas12-based type V, and Cas13 RNA-guided targets with collateral activities used in diagnostics.

Learn how PAM sequences distinguish foreign DNA from the CRISPR locus, guiding Cas proteins to recognize protospacers and cleave viral DNA in bacterial defense.

CRISPR-Cas9 stands out for genome editing because it is fast, cheap, precise, and easy to use, requiring only one Cas9 protein and lacking collateral cutting activity.

Explore the components of the type II CRISPR-Cas9 system, including crRNA, tracrRNA, and Cas9, assembled as a guide RNA to search and cut target DNA with programmable specificity.

Homology directed repair uses a donor template with homology to precisely repair double-stranded breaks, with MRN end resection, RPA protection, RAD51 invasion, and Holliday junction resolution enabling knock-ins.

Explore Cas9 nickase, where paired nickases generate two single-strand breaks for high-fidelity double nicking with two guide RNAs, reducing off-target effects and enabling homology-directed repair.

Fuse catalytically inactive dCas9 to the FokI nuclease to form fCas9, requiring dual gRNA binding with a 15–25 bp spacer, achieving greatly reduced off-target activity and high specificity.

Engineered SpCas9 variants expand PAM recognition beyond NGG to NGCG, NGAG, or NGA, widening CRISPR targeting scope while reducing off-target effects with a D1135E mutation.

Compare Cas9 orthologs from Staphylococcus aureus, Neisseria meningitidis, and Campylobacter jejuni with SpCas9, noting SaCas9's NNGRRT PAM and KKHSaCas9 variant, and NmCas9 and CjCas9 PAMs.

Explore how viral vectors deliver CRISPR components, such as gRNA and Cas9, into target cells via packaging cell lines, with integrating and non-integrating designs.

Explore adenoviral vectors entering cells via CAR receptor and delivering episomal DNA. Trace first to third generation vectors, including E1/E3 deletions, Ψ packaging signals, and transgene delivery such as Cas9.

Co-express CRISPR-Cas9 and site-specific gRNA in target cells to enable gene editing, using either an all-in-one adenoviral vector or separate vectors with co-transduction challenges.

Describe lentivirus-mediated CRISPR-Cas9 editing using gRNA and Cas9 transgenes in a vector, and how non-integrating lentiviral vectors mutate integrase to prevent random integration.

Prime editing offers greater precision and efficiency by targeting specific nucleotides without double-stranded DNA breaks, addressing delivery and off-target challenges of traditional CRISPR-Cas systems.

Identify prime editing components: Cas9 nickase fused to reverse transcriptase and pegRNA, which carries PBS and template RNA to enable targeted insertions, deletions, or base changes without double-strand breaks.

Prime editing uses a pegRNA and Cas9 nickase to introduce edits via a 3' DNA flap. The edited strand guides mismatch repair to permanently install changes in the genome.

Prime editing offers greater precision than CRISPR-Cas9 by using single-stranded breaks and DNA mismatch repair, but its large construct size limits delivery.

Base editing uses CRISPR-Cas to edit DNA precisely without double-strand breaks, via cytidine editors converting CG to TA and adenine editors converting AT to GC, guided by sgRNA.

Base editing provides precision with lower INDEL rates, enabling targeted insertion of beneficial mutations and showing potential for APOE4 and TP53 models as well as crop trait improvement.

Activate gene expression with the dCas9-VPR CRISPRa system, fusing VP64, p65, and Rta to dCas9 in an all-in-one vector with U6 gRNA sites and a CMV promoter-driven expression cassette.

Explore the Supernova Tagging CRISPRa system that links dCas9 to SunTag with GCN4 repeats to recruit VP64 antibodies, using three vectors to amplify gene expression by over 50-fold.

CRISPRi uses dCas9 to repress transcription by blocking RNA polymerase access when guided by gRNA, with enhanced repression from dCas9-KRAB fusion.

Explore engineered dna-binding molecule-mediated chip (enChIP) that uses catalytically inactive dCas9 with FLAG or BirA to identify proteins at a genomic locus via immunoprecipitation or proximity labeling and mass spectrometry.