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CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas Nuclease

Prokaryotic CRISPR array-Cas Nuclease (CRISPR/Cas) is an adaptive RNA-guided anti-[phage, virus, plasmid, transposon, integrative/conjugative elements, genomic island] defense system. CRISPR/Cas systems are capable of supporting mammalian gene editing (genetic engineering) applications by virtue of rational CRISPR RNA (crRNA/∼20 bp) sequence design coupled with exogenous genetically modifiable Cas Nuclease expression.

The human GeCKO (Genome-Scale CRISPR Knock-Out) libraries target early consecutive exons for genome editing.

CRISPR Editing Evaluation

Class 2 CRISPR Systems


Cas9 is an RNA guided DNA double strand endonuclease, that generates blunt end double strand DNA cuts via sgRNA targeting genomic DNA.

Type II CRISPR / RNA guided DNA Endonuclease / blunt end double strand DNA cut

  • ~64-nt guide RNA to encode target specificity.
  • Chimeric crRNA-tracrRNA hybrids (sgRNA) direct Cas9 cleavage within mammalian genomes to stimulate NHEJ or HDR-mediated genome editing.
  • Blunt ends; protospacer-adjacent motif (PAM) immediately downstream (3') of the target site.
  • saCas9 : 1053 aa Staphylococcus Aureus Cas9. PAM = NNGRRT. blunt end dsDNA cut
  • spCas9 : 1368 aa Streptococcus Pyogenes Cas9. PAM = N20-NGG. Versatile balance between PAM complexity (specificity) and construct size.
  • stCas9 : 1121 aa Streptococcus thermophilus (St1Cas9) PAM = NNAGAAW. 1388 aa St3Cas9 PAM = NGGNG


  • (1) DSB recognition
  • (2) processing of nonligatable DNA termini
  • (3) joining of two suitable DSBs
  • NHEJ constrains broken DNA ends in the absence of end resection for repair initiation (ie Insertion/Deletion (Indel)).
  • NHEJ : Ku heterodimeric repair scaffolds associate with DSB ends. Stochastic insertions and deletions (Indel) introduction occurs when complimentary strands undergo micro-alignment dependent end resection / repair misalignment leading to frame shift. Useful for gene disrutpion. Observable within 12 hours at high efficiencies (<70%).
  • Indel : Insertion deletion of bases in the genome of a cell.


HDR Plasmid can undergo cotransfection with Cas9/KO plasmid(s), and facilitates hereditary antibiotic-dependent cytotoxic selection of cells with genomic integration at Cas9-induced DNA cleavage sites. Homology Directed Recomination (HDR)/integration of an RFP/Puromycin resistance cassette.

  • Overview
  • Rad51 family association with DSBs recruit accessory factors directing genomic recombination via homologous arms on an exogenous repair template.
  • PCR Strategies for Cas9/HDR Homology Directed Repair HDR genomic integration variability
HDR enhancers
  • L-755,507 (CAS 159182-43-1) is a selective β3-adrenergic receptor partial agonist
  • L755507 and Brefeldin A, achieve maximal effects at 5 μM and 0.1 μM respective; increases efficiency of HDR dependent GFP insertion ~3 fold vs. DMSO-treated. L755507 increases HDR efficiency in cell lines (K562 and HeLa), suspension cells (K562), primary neonatal cells (HUVEC and fibroblast CRL-2097), and human ES cell-derived cells (neural stem cells)
  • (Li et al. 2011) Mechanistic by which L755507 enhances CRISPR-mediated HDR.
  • Treating different types of cells with L755507 shows improved HDR efficiency, ~2 fold in human umbilical vein endothelial cells (HUVEC). PMID: 25658371

Cas9 Nickase

Wild-type Cas9 Nuclease cleaves DNA via RuvC and HNH nuclease domains, that generate blunt end double strand breaks. Double nicking is achievable when Cas9 nickase (RuvC / HNH) complexing with appropriately spaced target sites mimic a double strand break via cooperative nicks.

  • Nickase HNH+  : HNH single nuclease domain cleaves DNA strand complementary to the RNA guide. Type 2 Streptococcus Cas9 contains 2 nuclease domains; RuvC / HNH
  • Nickase RuvC+ : RuvC 3 subdomain (RuvC1 @ N-terminal, RuvC2/3 flank HNH). Nuclease cleaves DNA strand non-complementary to the guide RNA. Type 2 Streptococcus Cas9 contains 2 nuclease domains; RuvC / HNH


  • dCas9-SAM: NLS-dCas9-VP64, and MS2-p65-HSF1 trans-acting native gene synergistic activation mediator (SAM).
  • dCas9-SSAP: Single-strand annealing protein(SSAP) dependent cleavage-free kilobase-scale (long-sequence genome engineering) gene editor (insertion).


  • U6 promoter:human
  • T2A peptide:Thosea asigna virus
  • GFP:Aequorea victoria
  • NLS:SV40 virus
  • Cas9:Streptococcus pyogenes bacteria
  • CBh promoter:chicken
  • gRNA scaffold:bacteria
  • 20 nt non-coding RNA sequence


  • EF1a Promoter:human
  • RFP:sea anemone Entacmaea
  • Puromycin:bacteria Streptomyces alboniger
  • T2A peptide:Thosea asigna virus

Cre Recombinase

  • Promoter:CMV virus
  • Cre:bacteriophage
  • NLS:SV40 virus


Type V CRISPR / RNA guided DNA Endonuclease / generates staggered cuts with 5'overhang

  • Cas12a: higher specificity/ lower seed mismatch tolerance (staggered dsDNA cut) versus Cas9 (blunt end dsDNA cut).
  • Acidaminococcus sp. BV3L6 Cas12a (AsCas12a, AsCpf1). 5′-TTTN-3′ PAM / staggered cut @ [~19bp downstream PAM (sense)/~23bp downstream PAM (antisense)].
  • Lachnospiraceae Cas12a (LbCas12a, LbCpf1).
  • Francisella novicida Cas12a (FnoCas12a).
  • Edits AT-rich genomes/regions/ SNP-specific editing.
  • Only requires crRNA (tracrRNA-independent)/processes crRNA without tracrRNA.
  • Dna Endonuclease-TargEted Crispr TRansreporter (DETECTR) achieves attomole DNA detection merging isothermal amplification with Cas12a.

DETECTR Microbiology Diagnostic

  • 1) RNA extracted from a sample
  • 2) reverse transcription (2&3 are in 1 reaction)
  • 3) isothermal amplification using loop-mediated amplification (RT–LAMP)
  • 4) pathogen sequence targeting gRNA dependent Cas12 detection
  • 5) Activated Cas12 dependent cleavage of a reporter molecule confirms positive detection


Type VI CRISPR / RNA guided RNA Endonuclease / permissive 'collateral' RNA cleavage

  • Programmable RNase activity= spacer complementarity-independent RNA/transcript degradation.
  • ~64-nt guide RNA scaffold for RNA endonuclease activation/ target specificity.
  • Target specificity via 28-30-nt spacer complementarity to target RNA.
  • Collateral cleavage
  • Collaborative post-recognition (non-specific) degradation of surrounding transcripts is limited to bacteria / not present in plant or mammal
  • crRNA spacer ~24 nt stem-loop structure with a mononucleotide protospacer
  • Specific High-sensitivity Enzymatic Reporter unLOCKing SHERLOCK Microbiology Diagnostic

Cas13a (C2c2)

Cas13b (C2c6)


  • Metabolizes endogenous transcript splicing
  • Possibile in vivo delivery due small size (~ 930 aa) /smallest size for class 2 CRISPR.


RNA-protein scaffold mapping


  • CBh  : chicken β-actin (CBA) hybrid promoter
  • CMV  : human cytomegalovirus (CMV) promoter regulatory drives constitutive protein expression; prone to methylation/silencing (ie iPSC)
  • DSB  : DNA double strand breaks undergo repair via error prone non-homologous end joining (NHEJ) or precision homology directed repair (HDR).
  • EF1A  : EF-1α promoter eukaryotic translation elongation factor 1 α (EF-1α, gene symbol EEF1A1) promoter increases transfection efficiency, the transgene expression, the proportion of expression-positive clones and in vitro copy number. Core EF1a promoter ~200-300bp in size. The ~1172bp intron-containing version (occasionally spliced out); the extra sequence yields higher level of expression than the core EF1a promoter alone.
  • HNH  : Cas9 Nuclease lobe (1 of 2); single nuclease domain.
  • HDR  : Rad51 family association with DSBs recruit accessory factors directing genomic recombination via homologous arms on an exogenous repair template.
  • Indel  : Insertion deletion of bases in the genome of a cell.
  • NHEJ  : Ku heterodimeric repair scaffolds associate with DSB ends. Stochastic insertions and deletions (Indel) introduction occurs when complimentary strands undergo micro-alignment dependent end resection / repair misalignment leading to frame shift. Useful for gene disrutpion. Observable within 12 hours at high efficiencies (<70%).
  • NUC  : Nuclease (NUC) lobe consisting of HNH domain, assembled RuvC subdomains, and a PAM-interacting (PI) C-terminal region.
  • PAM  : Target DNA sequence (protospacer) must be gRNA complimentary and contain a "protospacer adjacent motif" DNA sequence. PAM recognition -> rate-limiting R-loop propagation -> DNA dsDNA cleavage. protospacer adjacent motif (PAM) = ~2–6-base pair DNA sequence 3' to target DNA sequence of the RNA guided Cas9 nuclease (CRISPR bacterial adaptive immune system).
  • REC  : α-helical recognition (REC) lobe
  • RGN  : RNA guided Nuclease
  • RuvC  : Cas9 Nuclease lobe (2 of 2) RuvC contains three subdomains; RuvC I near the N-terminal region of Cas9 and RuvC II/III flanking the HNH domain near the middle of the protein.
  • sgRNA  : Fusion of ~20nt CRISPR RNA (crRNA) + ~60nt trans-activating RNA (tracRNA) simplifies RNA:Cas9 complex ratio from 2:1 -> 1:1, and improves editing rates relative to a dual RNA strategy. Targeted deletion utilizing 2+ sgRNA result in repair naive junctions by either sgRNA alone =higher % of Indel events. 1 sgRNA blunt end DSBs repair correct at a higher fidelity than 2+ sgRNA DSBs repair.
  • Seed Region: A subset of nucleotides within the crRNA (sgRNA) that pairs with PAM-proximal nucleotides; sensitive to mismatches, necessary for target affinity and specificity. Rate of genomic target binding (not affinity) decreases with each mismatch; CRISPRcas12 crRNA contains a larger seed region (lower mismatch tolerance) versus CRISPRcas9 (∼10 bp of the RNA-DNA helix (R-loop) proximal to the PAM).
  • Termination Signal: Poly TTTTTT will generate a "poly A tail" on nascent mRNA. Polyadenylation (poly(A)) signals (PAS) are a defining feature of eukaryotic protein-coding genes. The poly(A) tail is important for the nuclear export, translation and stability of mRNA.
  • U6: U6 is a type III RNA polymerase III promoter commonly used for driving ncRNA expression.




Cycle Threshold (cT) are inversely proportional to the amount of target nucleic acid in the sample (ie the lower the cT = the greater the amount of target nucleic acid)

  • cT = <29 = strong positive reactions / Abundant level of target primer amplicon nucleic acid (mRNA)
  • cT = 30-37 = positive reactions / Moderate level of target primer amplicon nucleic acid (mRNA)
  • cT = 38-40 = weak reactions / Minimum level of target primer amplicon nucleic acid (mRNA)

puromycin N-acetyltransferase


RFP (Red Fluorescent Protein)

RFP (DsRed)

RFP excites @488 nm laser/emits 585+/-8nm (similar to PE;565nm/575nm). Compatible multichannel fluorochromes with RFP = FITC, APC, PerCP-Cy5.5, PE-Cy7


GFP (Green Fluorescent Protein)


Synergistic Activation Mediator

To initiate programmable DNA targeting, dCas9 coordinates RNA-duplex (crRNA+tracrRNA) scaffold. crRNA nucleotides 1-20 guide dCas9 adjacent to genomic protospacer adjacent motif(s) (PAM). Fusion of crRNA+tracrRNA generates a chimeric single guide (sgRNA).

Synergistic activation mediator (SAM) system

  • Incorporates two MS2 hairpins into exposed loops within the sgRNA
  • Fuses MCP to a novel chimeric activator p65-HSF1 (p65 subunit of NF-kB/HSF1 heat shock factor 1 transcription response to temperature stress)
  • Chimeric MCP-p65-HSF1 activators bind each MS2 hairpin as a set of homodimers
  • 4 copies of MCP-p65-HSF1 scaffolding onto the SAM sgRNA, bound via dSpCas9-VP64
  • Optimal upstream (-200)-(+1) >> (-400)-(-50)


  • Recruitment of activators in trans
  • Insertion of protein binding RNA Aptamers (MS2) into sgRNA coordinates MCP
  • Heterogenous activator(s) (p65-HSF1)
  • Optimal upstream (-400)<->(-50) / (-200)<->(+1)

CRISPR Synergistic Activation Mediator Nomenclature

  • 2A  : self-cleaving peptide motif 2A peptide
  • dCas9: mutant Streptococcus (pyogenes) Cas9 nuclease with inactive catalytic domains utilized as a generic RNA-guided trans-modulator. RNA-guided DNA binding protein (dCas9). dCas9 may protect internal MS2 stemloops from exonuclease degradation.
  • HSF1 : Heat Shock Factor 1 transcribes gene in response to heat stress.
  • MCP  : MS2 Coat Protein (MCP) dimer binds each MS2 aptamer hairpin (1:1). (native MS2 system) Phage capsid assembly is nucleated by coat protein dimer binding to the operator hairpin.
  • MS2  : Escherichia virus (Enterobacteria phage) MS2 RNA "operator/aptamer hairpin". (tandem repeat fusion to 3' sgRNA). noncoding scaffold. minimal hairpin aptamer selectively binds dimerized MS2 bacteriophage coat protein (MCP). MS2 stem-loop placement within the sgRNA influences transcription activation efficiency.
  • p65  : NF-κB subunit. NF-κB trans-activating subunit p65 shares common co-factors with VP64, recruits a distinct subset of transcription factors and chromatin remodeling complexes.
  • RTA  : Human herpes virus 8 (HHV-8) transactivator.
  • sgRNA: single guide RNA = chimeric Crispr RNA (crRNA) + transactivating crRNA (tracrRNA).
  • VP64 : VP16 x4 / Herpes (alpha-TIF (trans-inducing factor) HHV-2 transactivator protein recruits transcription factors OCT-1 and Host cell factor 1 (HCF). (component of chimeric VP64-dCAS9).

Stem Cell / Sensitive Cell DNA Transfection

For DNA transfection of primary cells and sensitive cell lines, Effectene Transfection Reagent is a nonliposomal lipid reagent for DNA transfection into a broad range of cell types. Due to low cytotoxicity, Effectene Transfection Reagent is suitable on primary or other sensitive lineages.


Synergistic Activation Mediator


  • 13ul (5,000 particle/ µL) @ 200,000 cells per 35mm diameter = MOI 0.325
  • 6 Well / 10e5 (100,000 cells) per well /

200uL @5,000 particles/µL = 10e6 particles

MOI 1 = 20µL

MOI 3 = 60µL

MOI 5 = 100µL


  • Puromycin  : 0.5 µg/ml
  • Hygromycin B  : 75-100 µg/ml


  • Blasticidin S HCL : 0.5 µg/ml



【Lenti dCas9-VP64-Blast】

  • dCas9 (D10A/H840A) (Streptococcus pyogenes)
  • NLS: nuclear localization signal (SV40)
  • VP64 (Herpes Simplex Virus derived)
  • 2A peptide—>(Thosea asigna virus)
  • Blastcidin resistance gene—>Bacillus cereus
  • EF1a promoter—> human

【Lenti MS2-P65-HSF1-Hygro】

  • MS2:Bacteriophage MS2 coat protein
  • P65:Human
  • HSF1:Human
  • NLS: nuclear localization signal: (SV40)
  • 2A peptide:Thosea asigna virus
  • Hygromycin resistance gene:E,coli
  • EF1a promoter:human

【Lenti sgRNA (MS2)-Puro】

  • sgRNA
  • gRNA Scaffold :(MS2 aptamer-modified tetraloop and stem loop 2 (MS2 loops))
  • U6 promoter:human
  • Puromycin resistance gene:Streptomyces alboniger
  • EF1a promoter:human

HIV derived

  • 5'LTR: 5'Long Terminal Repeat (Lentivirus)
  • cPPT: Central Polypurine Tract (Lentivirus)
  • RRE: Rev Response Element (Lentivirus)
  • Psi Sequence (Lentivirus)
  • 3'LTR: 3'Long Terminal Repeat (Lentivirus)


Intersectional genetics

  • Flp (recombinase flippase (Flp))
  • 38kDa Cre (integrase) recombinase: topoisomerase I-like site specific recombination between two DNA recognition sites (LoxP sites; 34bp: 13 bp palindromic sequences flanking 8bp spacer)
  • type specific expression of target genes (EF1-LSL-tTA knockin) Eef1a1-2xloxP (LSL)-tetracycline-controlled transactivator (tTA). homologous recombination approach targeted insertion of (tTA) into endogenous eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) locus; tTA is preceded by a transcriptional Stop sequence (i.e., three polyA signals), which is surrounded by two loxP sites (LSL).
  • Dre/Cre recombinase

(Retro)Transposable elements


  • Exchange of replicating genetic information;recombination in DNA repair (ie repair of double-stranded breaks DSBs)