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CRISPR-Cas9

CRISPR-Cas9

CRISPR-Cas9 is a genome editing system. CRISPR systems provides immunity to bacteria and archaea from viruses and has been adapted for use as a genome editing tool capable of knocking out genes and rewriting genetic sequences in animal, plant and fungi. CRISPR-Cas9 is being adapted to other applications outside genome editing.

CRISPR-Cas9 is a genome editing system. The system originates in bacteria providing immunity to viruses and has been adapted for use as a genome editing tool capable of knocking out genes and rewriting genetic sequences in animal, plant and fungi. Outside of genome editing, modifications to the CRISPR-Cas9 system make it useful for gene regulation, genome imaging and studying protein-genome interactions.

History

Cas9 is the nuclease enzyme that does the cutting in the Type II CRISPR systems used by Streptococcus thermophilis . The function of Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) sequences that are part of a bacterial immune system, was discovered in the yogurt bacteria Streptococcus thermophilus . Phillipe Horvath and Rodolphe Barrangou of Danisco (later DuPont) made that discovery and in 2007 reported in Science that the bacteria incorporate sequences from phage viruses they have been exposed to as spacers in the CRISPR region which gave the bacteria resistance to those phage viruses . DuPont has patented a technique of exposing bacteria to different phage viruses and uses CRISPR sequences to tell them which ones have acquired resistance, something that helps them avoid phage viruses spoiling their yogurt .

Horvath and Barrangou teamed up with Virginjus Siksnys at Vilnius University, Lithuania and they published how CRISPR works with Cas9 in 2012 . Around the same time UC Berkeley’s Jennifer Doudna and Emmanuelle Charpentier now at the Max Planck Institute for Infection Biology, Berlin also described in Science how Cas9 works in the CRISPR system . Because they also engineered a simpler version of CRISPR that could likely to work in other organisms including human cells , this transformed this bacterial immune function into a usable biotechnology tool.

There is a patent dispute over the invention of CRISPR-Cas9 technology specifically for use in human cells, between Doudna’s research team and Feng Zhang’s group at the Broad Institute (MIT and Harvard) . Zhang’s research group and George Church’s lab at Harvard Medical School, each published Science papers in 2013 showing they had modified CRISPR-Cas9 to edit the genome in human and mouse cells . The Broad Institute’s US patent, the first of several for mammalian use of CRISPR, is under appeal . Citing lack of novelty, the European patent office has revoked the first patent obtained by the Broad Institute and has granted patents to the University of California and University of Vienna . The first is for using the CRISPR-Cas9 system across prokaryotic and eukaryotic systems and the second is for a modified form of CRISPR-Cas9 to regulate gene expression.

Genome Editing

Targeting of Cas9 to cleave DNA in bacterial immune function uses two RNAs which form a duplex, the crRNA that recognizes the invading DNA and the tracrRNA which hybridizes with the crRNA . Doudna’s group engineered the system to use a single guide RNA . The CRISPR-Cas9 genome engineering system uses a single protein, Cas9, and single guide-RNA complex (Cas9-sgRNA) and it is the most commonly used CRISPR system for gene editing . The CRISPR-Cas9 user designed guide RNA binds DNA that contains the complementary sequence. The presence of a nearby (protospacer-adjacent motif) PAM sequence is required for cleavage in the target region. For endogenous CRISPR systems in bacteria, absence of PAM sequences in the bacteria’s own genome prevent self cleavage . In the human genome, the short PAM sequence is present at a frequency of 5.21% .

After CRISPR-Cas9 cleaves the DNA, the double stranded break triggers repair by either non-homologous end joining (NHEJ) or homology-directed repair (HDR) . NHEJ can knockout gene function due to random insertions or deletions occur at the site that disrupt the reading frame or change the protein coded for by the sequence. Researchers take advantage of the HDR mechanism by supplying a user-generated DNA template which is used to correct the break. In this way a gene mutation can be cut out and replaced with a corrected version of the gene. Scientists use CRISPR-Cas9 to alter genes in model organisms and cell lines to learn the function of those genes. CRISPR-Cas9 is also being used to develop therapies to treat or cure genetic diseases.

A modified form of CRISPR-Cas9 where dCas9 cannot cut DNA but maintains the ability to target DNA. When fused with transcriptional activators and repressors it can turn gene expression up or down. Similarly, Epigenomic CRISPR-Cas9 systems are fused to proteins that recruit epigenetic modifiers to a target genomic region, resulting in changes in gene expression.

Cancer

There are clinical trials at Hangzhou Cancer Hospital, China and in the US by University of Pennsylvania researchers where the human immune cells, T-cells are removed from patients, modified by CRISPR-Cas9 in a way that enhances their ability to fight cancer, and put back into the patients .

Blood Disorders

In collaboration CRISPR Therapeutics and Vertex Pharmaceuticals are using CRISPR-Cas9 outside the body to correct a genetic mutation in blood cells of patients with Beta-Thalassemia and Sickle Cell Disease . Clinical trials for these diseases using CRISPR have been put on hold in the US to resolve questions about safety .

Blindness

Editas Medicine is developing a CRISPR-Cas9 therapy for Leber Hereditary Amaurosis . Autosomal recessive and autosomal dominant Retinitis Pigmentosa, choroidal neovascularization and Age-Related Macular Degeneration are forms of blindness that are being tested with CRISPR-Cas9 preclinically in animal models .

Cystic Fibrosis

CRISPR can fix the Cystic Fibrosis (CF) mutation in lung cells, intestinal cells, and iPS cells derived from patients . Editas Medicine and CRISPR Therapeutics are working towards a therapy to use on CF patients .

Duchenne Muscular Dystrophy

Research in mice demonstrates CRISPR-Cas9 can fix Duchenne Muscular Dystropy (DMD) mutations . However since there are many mutations that cause the disease in humans and one in three are new mutations, it is a challenge to design a gene editing fix that works for more than just one mutation . Eric Olsen and his team at University of Texas Southwestern Medical Center developed a CRISPR-Cas9 gene editing technique that targets 3000 types of DMD mutations . Their system uses 12 guide RNAs that target mutation hotspots to restore heart muscle function in human heart tissue derived from patients . Their system causes changes splice sites so that most commonly mutated sections of the RNA coding for the protein are skipped, a strategy called exon-skipping or myoediting. The resulting protein while still incomplete is still able to function well enough.

Timeline

February 13, 2013
Zhang and Church each publish papers with CRISPR-Cas9 editing in human cells
August 17, 2012
Doudna and Charpentier describe how CRISPR works and engineer simpler form of CRISPR-Cas9
March 23, 2007
Barrangou and Horvath publish that bacteria use CRISPR to aquire resistance to viruses

People

Name
Role
LinkedIn

Emmanuelle Charpentier

Eric Olsen

Feng Zhang

George Church

Jennifer Doudna

Philippe Horvath

Rodolphe Barrangou

Virginjus Siksnys

Further reading

Title
Author
Link
Type
Date

A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi

Christina S. Nødvig, Jakob B. Nielsen, Martin E. Kogle, Uffe H. Mortensen

Journal

A CRISPR-Cas9-based gene drive platform for genetic interaction analysis in Candida albicans

Rebecca S Shapiro, Alejandro Chavez, Caroline B M Porter, Meagan Hamblin, Christian S Kaas, James E Dicarlo, Guisheng Zeng, Xiaoli Xu, Alexey V Revtovich, Natalia V Kirienko, Yue Wang, George M Church, James J Collins

Journal

CRISPR/Cas9 in Genome Editing and Beyond

Wang, H., La Russa, M. and Qi, L.S.

Safeguarding CRISPR-Cas9 gene drives in yeast

James E Dicarlo, Alejandro Chavez, Sven L Dietz, Kevin M Esvelt, George M Church

Journal

Xconomy: Pre-Existing Immunity to CRISPR Found in 96% of People in Study

Web

Documentaries, videos and podcasts

Title
Date
Link

TED - How CRISPR let's us edit our DNA | Jennifer Doudna

11/12/2015

Companies

Company
CEO
Location
Products/Services

Caribou Biosciences

CRISPR Therapeutics

Editas Medicine

ERS Genomics

Intellia Therapeutics

Vertex Pharmaceuticals

News

Title
Author
Date
Publisher
Description
Mark Terry
May 7, 2021
BioSpace
Every week there are numerous scientific studies published. Here's a look at some of the more interesting ones.
Science X staff
April 30, 2021
phys.org
While the CRISPR-Cas9 gene editing system has become the poster child for innovation in synthetic biology, it has some major limitations. CRISPR-Cas9 can be programmed to find and cut specific pieces of DNA, but editing the DNA to create desired mutations requires tricking the cell into using a new piece of DNA to repair the break. This bait-and-switch can be complicated to orchestrate, and can even be toxic to cells because Cas9 often cuts unintended, off-target sites as well.
Arlene Weintraub
April 30, 2021
FierceBiotech
Zebrafish can recover from spinal cord injuries, thanks to a healing process that's controlled by immune cells called macrophages. Researchers from the University of Edinburgh the gene editing technology CRISPR-Cas9 to identify four genes that are crucial for repairing severed spinal cords.
Karl Petri
April 29, 2021
Nature Biotechnology
Prime editors have been delivered using DNA or RNA vectors. Here we demonstrate prime editing with purified ribonucleoprotein complexes. We introduced somatic mutations in zebrafish embryos with frequencies as high as 30% and demonstrate germline transmission. We also observed unintended insertions, deletions and prime editing guide RNA (pegRNA) scaffold incorporations. In HEK293T and primary human T cells, prime editing with purified ribonucleoprotein complexes introduced desired edits with frequencies of up to 21 and 7.5%, respectively. Prime editors are delivered as ribonucleoproteins to zebrafish embryos and human primary cells.
Karl Petri
April 29, 2021
Nature Biotechnology
Prime editors have been delivered using DNA or RNA vectors. Here we demonstrate prime editing with purified ribonucleoprotein complexes. We introduced somatic mutations in zebrafish embryos with frequencies as high as 30% and demonstrate germline transmission. We also observed unintended insertions, deletions and prime editing guide RNA (pegRNA) scaffold incorporations. In HEK293T and primary human T cells, prime editing with purified ribonucleoprotein complexes introduced desired edits with frequencies of up to 21 and 7.5%, respectively. Prime editors are delivered as ribonucleoproteins to zebrafish embryos and human primary cells.
Noa Liscovitch-Brauer
April 29, 2021
Nature Biotechnology
CRISPR screens have been used to connect genetic perturbations with changes in gene expression and phenotypes. Here we describe a CRISPR-based, single-cell combinatorial indexing assay for transposase-accessible chromatin (CRISPR-sciATAC) to link genetic perturbations to genome-wide chromatin accessibility in a large number of cells. In human myelogenous leukemia cells, we apply CRISPR-sciATAC to target 105 chromatin-related genes, generating chromatin accessibility data for ~30,000 single cells. We correlate the loss of specific chromatin remodelers with changes in accessibility globally and at the binding sites of individual transcription factors (TFs). For example, we show that loss of the H3K27 methyltransferase EZH2 increases accessibility at heterochromatic regions involved in embryonic development and triggers expression of genes in the HOXA and HOXD clusters. At a subset of regulatory sites, we also analyze changes in nucleosome spacing following the loss of chromatin remodelers. CRISPR-sciATAC is a high-throughput, single-cell method for studying the effect of genetic perturbations on chromatin in normal and disease states. The effects of gene knockouts on chromatin accessibility are measured with single-cell CRISPR screens.
Shuai Jin
April 15, 2021
Nature Biotechnology
Although prime editors (PEs) have the potential to facilitate precise genome editing in therapeutic, agricultural and research applications, their specificity has not been comprehensively evaluated. To provide a systematic assessment in plants, we first examined the mismatch tolerance of PEs in plant cells and found that the editing frequency was influenced by the number and location of mismatches in the primer binding site and spacer of the prime editing guide RNA (pegRNA). Assessing the activity of 12 pegRNAs at 179 predicted off-target sites, we detected only low frequencies of off-target edits (0.00~0.23%). Whole-genome sequencing of 29 PE-treated rice plants confirmed that PEs do not induce genome-wide pegRNA-independent off-target single-nucleotide variants or small insertions/deletions. We also show that ectopic expression of the Moloney murine leukemia virus reverse transcriptase as part of the PE does not change retrotransposon copy number or telomere structure or cause insertion of pegRNA or messenger RNA sequences into the genome. A very high specificity of prime editors is demonstrated in a study of their off-target activity in plants.
Laura Spinney
March 11, 2021
the Guardian
Designer babies and ethical quicksand ... The biographer of Steve Jobs tells the story of Jennifer Doudna and the development of gene-editing
Laura Spinney
March 11, 2021
the Guardian
Designer babies and ethical quicksand ... The biographer of Steve Jobs tells the story of Jennifer Doudna and the development of gene-editing
Will Knight
March 8, 2021
Wired
The Nobel Prize-winning novelist Kazuo Ishiguro talks to WIRED about AI, CRISPR, and his hopes for humanity.
Philip Ball
February 21, 2021
the Guardian
Illustration by Paul Tansley.
Philip Ball
February 21, 2021
the Guardian
Illustration by Paul Tansley.
Research and Markets
February 9, 2021
www.prnewswire.com:443
/PRNewswire/ -- The "Global CRISPR Gene Editing Market: Focus on Products, Applications, End Users, Country Data (16 Countries), and Competitive Landscape -...
Special Correspondent
February 3, 2021
The Hindu
CRISPR-Cas9 technology set to revolutionise medicine and agriculture: Prof. Hefferon
Angus Liu
January 28, 2021
FierceBiotech
CRISPR-Cas9 has made waves in the biomedical world as a revolutionary gene editing tool, but it has its limitations. A team from the University of Illinois at Urbana-Champaign showed that an alternate technology, TALEN, is up to five times more efficient than CRISPR-Cas9 in highly compact forms of DNA.
Angus Liu
January 22, 2021
FierceBiotech
Cancer metastasis is responsible for most disease-related deaths, but it's hard to trace. Now, researchers led by MIT have developed a CRISPR-based method that helped them track the spreading patterns of tumor cells. What they learned could guide the development of new cancer drugs.
Angus Liu
January 22, 2021
FierceBiotech
Cancer metastasis is responsible for most disease-related deaths, but it's hard to trace. Now, researchers led by MIT have developed a CRISPR-based method that helped them track the spreading patterns of tumor cells. What they learned could guide the development of new cancer drugs.
Polaris Market Research
December 16, 2020
www.prnewswire.com:443
/PRNewswire/ -- The global CRISPR and Cas genes market is projected to reach USD 4.75 Billion By 2027, and is expected to grow at a compound annual growth rate...
Science X staff
December 10, 2020
phys.org
CRISPR-Cas9 makes it easy to knock out or tweak a single gene to determine its effect on an organism or cell, or even another gene. But what if you could perform several thousand experiments at once, using CRISPR to tweak every gene in the genome individually and quickly see the impact of each?
Science X staff
December 4, 2020
phys.org
A group of researchers developed a promising fix to CRISPR-Cas9's problem with unwanted genetic changes using a method that allows them to turn off gene-editing until it reaches key cell cycle phases where more accurate repairs are likely to happen.
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References

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