CRISPR

Genome Editing

changing the sequence of DNA, deleting pieces of DNA, adding pieces of DNA, genome editing with engineered nucleases (GEEN)

Transcription Activator-Like Effector Nucleases

custom designed TAL effector DNA binding domain attached to nuclease domain; secreted by Xanthomonas bacteria when they infect plants; DNA binding domain contains a highly conserved 33-34 amino acid sequence each recognizing one nucleotide

Zinc Finger Nucleases

zinc finger array is attached to FokI cleavage domain, zinc finger array attaches to half site on either end of spacer where DNA gets cut, use combination zinc finger domains to recognize DNA; each zinc finger recognizes 9-18 base pairs

CRISPR

Clustered Regularly Interspersed Short Palindromic Repeats; first described in E.Coli

E. Coli CRISPR

14 repeats of 29 bp interspersed by 32-33 bp non-repeating spacer sequences; similar structures form in 40% of bacteria and 90% of archaea; part of ‘bacterial immune system’

Cas Genes

Crisper associated genes

How CRISPR works

1. virus invades bacterial cell

2. new spacer derived from virus is integrated into CRISPR sequence

3. CRISPR RNA formed

4. CRISPR RNA guides molecular machinery to target and destroys viral genome

CRISPR Type II system

adaptation where a piece of foreign DNA - a protospacer - lies upstream of a protospacer adjacent motif (NGG, with N = any base); protospacer is then incorporated into bacterial genome at CRISPR Locus

production of crRNA

RNAase III and Cas9 line up tracrRNA with precursor cRNA by lining up repeat region; RNAase transcribes crRNA; makes a complex of tracrRNA, cas9, and crRNA which will line up to the genomic DNA and cut it with cas9

components of CRISPR-Cas9 system

Cas9 nuclease with RuvC and HNH, crRNA, tracrRNA

RuvC

endonuclease domain, cuts one strand of DNA

HNH

histidine asparagine histidine motif

What is needed to edit genome

Cas9, sgRNA (guide RNA) which is a combination of crRNA and tracrRNA, repair template (optional)

Limitations of CRISPR-Cas9

need protospacer adjacent motif (PAM); non-specificity (cas9 can cut genome at non targeted sites with low frequency); two component system; induction of DSB can have effects on cell fitness, CRISPR-sgRNA delivery methods are not great; editing efficiency is low

Gene Knockout

Cas9 cuts at a specific location disrupting a gene; NHEJ is used to repair double stranded break so it: returns to normal gene, insertion mutation, deletion mutation, or frameshift mutation

Gene Knock-in

cas9 cuts at a specific mutation, a repair template is provided with desired genomic edit, homology directed repair incorporates the genomic edit

non-editing applications of CRISPR

dCas9 has RUVC and HNH domain mutants creating a dead Cas9 and then can be applied in non-editing applications

gene activation with dcas9

an activator bound to cas9 binds upstream to activate a gene; dCas9+vP64 +SUN-Tay + P300; avoids DNA damage, can be used to manipulate non-promotor regions

gene repression with dCas9

dCas9 with a repressor bound binds upstream; dCas9+KRAB+EZH2+DNMT3A; can manipulate non promotor regions, avoids DNA damage

visualization using dCas9

dCas9 fuses with G-fluorescent protein which allows tracking of genomic locations over time, more easily adaptable than other genomic tracking methods; can be combined with different fluorescent markers for multiplexing

Cataracts treatment

Crygc mutations can cause cataracts, CRISPR editing to remain mutation with HDR alleviated cataracts in mice

Hutchinson-Guilford Progeria treatment

caused by a single base change in LMNA gene causing exon skipping and a 150 bp shorter mRNA, mouse model can have lifespan extended by CRISPR mediated editing of LMNA gene to restore exon II sequence

CASGEVY

treats B-thalassemia; first FDA approved gene therapy using CRISPR, targets cas9 to BCLIIA enhancer causing knock out of the enhancer, less BCLIIA expression means less mediated repression of HBF genes, which increases levels of HBF, and reduces mutant hemoglobin

Transthryetin amyloidosis

caused by misfolded TTR in hepatocytes; CRISPR disruption of TTR gene causes framshift mutation which lowers production of TTR and lessens symptoms

Type I CRISPR system

multiprotein crRNA effector module; cas3, ssDNA stimulated helicase often fused with an endonuclease

Type III CRISPR system

multiprotein effectors including Cas10 which has an RNA recognition sequence, often fused to an endonuclease

Type V CRISPR system

Cas 12

Type VI CRISPR system

cas13b, editing RNA bases directly with CRISPR