Cas9
Cas9 is a protein that in tandem with CRISPR can permanently modify a cell’s genome. It is a key tool of genetic engineering. Emmanuelle Charpentier and Jennifer Doudna won the 2020 Nobel Prize in Chemistry for developing the CRISPR genome editing technique.
Cas9 serves as a genome editing tool, inducing site-directed double-strand breaks in DNA, enabling gene inactivation or insertion via non-homologous end joining or homologous recombination. Variants like Cas9 nickase, which creates single-strand breaks, or those recognizing different PAM sequences, address limitations in CRISPR-Cas9 editing.
History
Mechanism
160-kilodalton Native Cas9 requires a guide RNA composed of two disparate RNAs that associate – the CRISPR RNA, and the trans-activating crRNA.Research
In 2013, Mali, et al., as well as Gilbert, et al., reported that non-cleaving Cas9 variants can control transcriptional activators or repressors at specific DNA sequences. Also in 2013, Bikard, et al., reported that dCas9 blocks RNA polymerase at promoters or during elongation, silencing genes.In 2014, Esvelt, Smidler, Catteruccia, and Church suggested that Cas9-based gene drives could edit entire organism populations.
2017-19
In 2017, Li, et al., reported that Cas9 targets Hepatitis B viral genome ends, reducing infection.Jensen et al., reported that fused to chromatin remodelers, dCas9 alters chromatin structure. O’Geen, et al., reported that dCas9 with FOG1 represses genes by methylating H3K27.
Lowder, et al., reported that dCas9 represses plant genes like AtCSTF64 when fused to repressor domains.
In 2018, Lee, et al., reported that engineered Cas9 reduces off-target effects by altering reaction rates.
In 2019, Chen and Page-McCaw reported that Cas9 suppresses HIV-1 long terminal repeats, mutating the viral genome.
2020
In 2020, Uddin, Rudin, and Sen reported that Cas9 variants, such as Cas9 nickase, improve genome editing by reducing off-target effects.2025-
In 2025, Zhou, Diao, Li, and colleagues reported that apoNmeCas9 can stimulate Cas1–Cas2-mediated CRISPR spacer acquisition in a type II-C system when CRISPR RNA levels are low.CRISPR-mediated immunity
CRISPR systems in bacteria and archaea act as programmable restriction enzymes to combat viruses and plasmids. CRISPR loci consist of short, palindromic repeats and variable spacers that store sequences from foreign DNA. Adjacent cas genes, including universal cas1 and cas2, and type II-specific cas9, encode proteins for immunity. Cas9 uses crRNA and tracrRNA to form a complex that targets and cleaves foreign DNA matching the spacer sequence.Adaptation
CRISPR systems integrate spacers from viral “protospacers” into the CRISPR locus, requiring a PAM for recognition. Cas9 ensures functional spacer acquisition. However, spacers can be lost via homologous recombination.ApoCas9 was reported to stimulate spacer acquisition in type II-C systems and to couple acquisition activity to CRISPR RNA abundance, with low crRNA associated with increased acquisition and higher crRNA associated with reduced acquisition. The nuclease lobe alone was sufficient to stimulate acquisition, whereas crRNA-dependent regulation required full-length Cas9; This activity is evolutionarily conserved across several type II-C Cas9 orthologues.
CRISPR processing
RNA polymerase transcribes the CRISPR locus into pre-crRNA, which specific endoribonucleases cleave into small crRNAs containing one spacer and a partial repeat.Interference
The crRNA, within a CASCADE complex, pairs with complementary foreign DNA. Cas9 cleaves the DNA if the spacer matches and the PAM is present, halting viral replication.Transcription regulation
Inactive Cas9, lacking endonuclease activity, binds DNA to regulate transcription.In eukaryotes, dCas9 targets enhancer sequences to prevent transcription factor assembly. Gilbert, Horlbeck, Adamson, Villalta, Chen, Whitehead, Guimaraes, Panning, Ploegh, Bassik, Qi, Kampmann, and Weissman conducted genome-wide dCas9 screens for gene repression. O’Connell, Oakes, Sternberg, East-Seletsky, Kaplan, and Doudna reported that Cas9 cleaves mRNA when hybridized with ssDNA containing a PAM complement. dCas9 fused to transcription activators, as in CRISPRa, activates genes, as reported by Gilbert et al.
Structural studies
Cas9 features a bi-lobed structure with an alpha-helical lobe and a nuclease lobe containing HNH and RuvC domains, connected by a bridge helix. The HNH domain cleaves the target DNA strand, while RuvC cleaves the non-target strand. The PAM-interacting domain recognizes the NGG PAM sequence. The sgRNA, replacing the crRNA-tracrRNA complex, forms a T-shaped structure with the target DNA. A 2014 study reported that REC1 and bridge helix domains are critical for sgRNA recognition.DNA cleavage
Cas9’s HNH and RuvC domains cleave the target and non-target DNA strands, respectively, requiring a PAM sequence. A 2020 study reported that NAG and NGA PAMs are less efficient. A 2016 study reported that cleavage often produces 1-nucleotide 5’ overhangs, favoring templated insertions. A 2024 study reported that 85% of on-target cleavages are blunt, while 15% have 1-nucleotide overhangs. A 2018 study reported that cleavage efficiency depends on free energy changes in the gRNA-DNA duplex. Another 2016 study reported that stable gRNA folding can impair cleavage.Challenges in bacterial editing
A 1995 study reported that bacterial restriction-modification systems cleave foreign DNA, including Cas9-introduced genes, hindering genome editing.Applications
Gene editing
Cas9 edits genomes in a range of species, including yeast, Candida albicans, zebrafish, fruit flies, ants, mosquitoes, nematodes, plants, mice, and monkeys.Cellular defense
CRISPR/Cas9 can help with bacterial defense against invaders such as bacteriophages, plasmids, and DNA viruses. Cas9, guided by RNA, acts as an endonuclease enzyme, targeting and cleaving foreign DNA. It unwinds foreign DNA and cleaves matching sites, similar to the RNA interference mechanism in eukaryotes.Gene regulation
Other versions of Cas9 can bind but not cleave DNA. They can be used to control transcriptional activator or repressor genes and manage gene activation and repression.More technically, Cas9 is a RNA-guided DNA endonuclease enzyme associated with the Clustered Regularly Interspaced Short Palindromic Repeats adaptive immune system in Streptococcus pyogenes. S. pyogenes utilizes CRISPR to memorize and Cas9 to later interrogate and cleave foreign DNA, such as invading bacteriophage DNA or plasmid DNA. Cas9 performs this interrogation by unwinding foreign DNA and checking for sites complementary to the 20 nucleotide spacer region of the guide RNA. If the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA. In this sense, the CRISPR-Cas9 mechanism parallels the RNA interference mechanism in eukaryotes.
Apart from its original function in bacterial immunity, Cas9 has been utilized as a genome engineering tool to induce site-directed double-strand breaks in DNA. These breaks can lead to gene inactivation or the introduction of heterologous genes through non-homologous end joining and homologous recombination respectively in model organisms. Research on cas9 variants has been a promising way of overcoming the limitation of the CRISPR-Cas9 genome editing. Examples include Cas9 nickase, a variant that induces single-stranded breaks or variants recognizing different PAM sequences. Alongside zinc finger nucleases and transcription activator-like effector nuclease proteins, Cas9 is becoming a prominent tool in the field of genome editing.
Cas9 has gained traction in recent years because it can cleave nearly any sequence complementary to the guide RNA. Because the target specificity of Cas9 stems from the guide RNA:DNA complementarity and not modifications to the protein itself, engineering Cas9 to target new DNA is straightforward. Cas9 targeting has been simplified through the engineering of a chimeric single guide RNA. Scientists have suggested that Cas9-based gene drives may be capable of editing the genomes of entire populations of organisms. In 2015, Cas9 was used to modify the genome of human embryos for the first time.
History
The Cas9 endonuclease is a four-component system that includes two small molecules: crRNA and trans-activating CRISPR RNA. In 2012, Jennifer Doudna and Emmanuelle Charpentier re-engineered the Cas9 endonuclease into a more manageable two-component system by fusing the two RNA molecules into a "single-guide RNA" that, when combined with Cas9, could find and cut the DNA target specified by the guide RNA. This contribution was so significant that it was recognized by the Nobel Prize in Chemistry in 2020. By manipulating the nucleotide sequence of the guide RNA, the artificial Cas9 system could be programmed to target any DNA sequence for separation. Another collaboration comprising Virginijus Šikšnys, Gasiūnas, Barrangou, and Horvath showed that Cas9 from the S. thermophilus CRISPR system can also be reprogrammed to target a site of their choosing by changing the sequence of its crRNA. These advances fueled efforts to edit genomes with the modified CRISPR-Cas9 system.Groups led by Feng Zhang and George Church simultaneously published descriptions of genome editing in human cell cultures using CRISPR-Cas9 for the first time. It has since been used in a wide range of organisms, including baker's yeast, the opportunistic pathogen Candida albicans, zebrafish, fruit flies, ants, mosquitoes, nematodes, plants, mice '', monkeys and human embryos.
CRISPR has been modified to make programmable transcription factors that allow activation or silencing of targeted genes.
File:Cas12a vs Cas9 cleavage position.svg|thumb|A diagram of the CRISPR nucleases Cas12a and Cas9 with the position of DNA cleavage shown relative to their PAM sequences in a zoom-in
The CRISPR-Cas9 system has been shown to make effective gene edits in Human tripronuclear zygotes, as first described in a 2015 paper by Chinese scientists P. Liang and Y. Xu. The system made a successful cleavage of mutant Beta-Hemoglobin in 28 out of 54 embryos. Four out of the 28 embryos were successfully recombined using a donor template. The scientists showed that during DNA recombination of the cleaved strand, the homologous endogenous sequence HBD competes with the exogenous donor template. DNA repair in human embryos is much more complicated and particular than in derived stem cells.