... single mutagenic primer pair to generate an individual mutant, this technique uses mutagenic primers ...2nd mutant strand: The second mutant strand is synthesized in the same manner as the first mutant strand...Pooled Libraries to Generate Mutant Populations Use REPLACER Mutagenesis to Mutate Your Plasmids Resources...libraries of mutant proteins. Researchers from the Whitehead lab developed One pot saturation mutagenesis as a...generate complex libraries of mutant plasmids ready for deep mutational scanning.     3 reasons to be...to one pot saturation mutagenesis Several methods can be used to create mutational scanning libraries. ...favoring of certain types of mutations, difficulty controlling the rate of mutagenesis, and low throughput. ...

...directed mutagenesis to generate specific mutations in your plasmids Use REPLACER Mutagenesis to mutate your... to mutagenesis with error prone PCR, EvolvR uses an error prone polymerase to introduce mutations. However... 15-20 bp. EvolvR’s on-target mutagenesis rate was 2.5 x 10-6 mutations per nucleotide per generation ...increase the mutation rate and extend the editing window of EvolvR: (1) two additional mutations to PolI3M... PolI5M which increased EvolvR’s mutagenesis rate to ~10-3 mutations per nucleotide per generation, and...many known spectinomycin resistance mutations, several new mutations were identified by targeting EvolvR...Mutagenesis is a tool that both evolution and molecular biologists use to tinker with DNA. Making changes...

... the mutagenesis reaction. Alternative approaches to site directed mutagenesis Point-mutagenesis is fairly...designed mutations (not limited to frame shift mutations). Consequently, site directed mutagenesis is one...contains the template, site-directed mutagenesis can be used to mutate the PAM sequence (an NGG sequence ...cleavage. Summary of Site Directed Mutagenesis In brief, point-mutations can be introduced to plasmids using...  Site-directed mutagenesis of Plasmids. A) Generation of mutagenized plasmids. PCR primers (green...through mutagenesis vastly facilitates the subsequent process of screening for succesfully mutated clones...site directed mutagenesis.  Polymerase To ensure that you don’t introduce undesired mutations through the...

... point mutations Insertions, deletions, point mutations Insertions, deletions, point mutations Insertions...inactivating/activating mutations. In biomedical research, modeling patient mutations using SDM can help determine...directed mutagenesis methods Current SDM methods rely on in vitro assembly. To make a point mutation, you ...desired mutation, then use those to PCR amplify the template plasmid. The parental, nonmutated DNA is ...REPLACR-mutagenesis is that you must PCR amplify the whole plasmid, which could introduce mutations. Using...bp homology, the observed efficiency of mutagenesis (mutation-containing colonies/total colonies) is about...REPLACR-mutagenesis could save busy scientists a lot of time. Let us know how REPLACR-mutagenesis works ...

...attempting to characterize mutations in a pooled cell population, as mutations with strong phenotypic effects...types of mutations they identified included gene amplifications and fusions, missense mutations, and indels...Functional Annotation of Somatic Mutations in Cancer.” Of all the mutations and fusions represented in this...their associated mutants in other model systems. It is possible that a given mutation will have a different...Thousands of mutations have been implicated in cancer. Yet, the majority of them remain uncharacterized...first functional characterization of over 1,000 mutations, and the group has deposited several hundred plasmids...assortment of genes. Using patient data to identify mutations of unknown importance in cancer Previously available...

...characterized by deficient phosphomannomutase (PMM) activity due to mutations in the PMM2 gene. Videira...diseases. Spontaneous mutations are a major cause of rare diseases. Spontaneous mutations that change protein...diseases caused by Arg-mutant proteins, but there is currently no evidence of Arg-mutant protein rescue in ...rescue multiple Arg-mutant proteins in living baker’s yeast cells, including Arg-mutant yeast homologs of...research on diseases of glycosylation, arginine mutagenesis, and neural development. Paula Videira: Congenital...27393411. Michael McMurray: Rescuing arginine mutations in rare disease with guanidine Michael McMurray...other. Indeed, nearly 20% of the single missense mutant alleles that cause human disease change Arg to ...

...blocking mutation. Despite the fact that both the blocking mutation and our intended mutation were on ...or heterozygous mutations. 1. Increasing HDR accuracy with CRISPR/Cas-blocking mutations After genome editing...sequence. The idea was that these “blocking mutations” mutations prevent CRISPR from re-cutting the target...blocking mutations? This depends entirely on the locus you are editing, and if the blocking mutation needs...intended mutation while taking advantage of the improved efficiency provided by blocking mutations. Essentially...cut site to mutation distance and editing efficiency.   3. Optimizing "cut-to-mutation distance" for... Alzheimer mutations we study are heterozygous in patients. As the probability of mutation incorporation...

...Surveyor® or T7E1) for mutation detection. While these methods paved the way for mutation validation, we found...and is not capable of binding a mutated target. The amount of both mutated and wild type target in each ...samples with mono-allelic mutations versus those with the bi-allelic mutations typically desired when screening... comes time to (hopefully) validate successful mutation of your favourite gene, there are several different... to help guide you through the rough waters of mutation validation, such as this "CRISPR 101" post. Next-generation... molecule in a sample. Digital PCR assays for mutation screening Now that we have introduced how digital... assays can be used to sniff out your desired mutations. The assays you will use aren’t that much different...

...uptake, iron uptake receptor mutation. T1/T5 Phage resistance gal Mutation in galactose metabolism pathway...DNA, preparing unmethylated DNA, and more. Many mutations that make these features possible are present ...present in most commercial strains, especially mutations that make major improvements such as those that increase...found in E. coli strains. Table 1: Common gene mutations found in E.coli strains Gene(s) Description...Functional Consequence dam DNA adenine methylase mutation (GATC) Preparing unmethylated DNA, important when...ex: ClaI or XbaI) dcm DNA cytosine methylase mutation (CCWGG) Preparing unmethylated DNA, important ...enzymes that are methylation sensitive. dnaJ Mutation in a chaperonin gene Increases the stability of...

...commonly used to introduce multiple mutations. This is because as more mutations are introduced into a single...arise when crossing mice with multiple mutations is if the mutations are on the same chromosome. Homologous...engineering should be used to separately introduce each mutation independently into different mice, and then these...these strains should be crossed to generate double-mutant offspring. Crossing mice allows for the production...production of offspring that have a combination of the mutations engineered into each of their parents. Here’s ...mice that are each homozygous for one of the two mutations that you want combined in your offspring. Genotyping...producing mice with your desired combination of mutations. Assuming your parental mice were all homozygous...

...to combine the best of these mutations and tested the combination mutants for their on-target and off-.... showed that these mutants are not toxic to HEK cells and that similar mutations can improve the specificity...strand via mutations N497A, R661A, Q695A, and Q926A (SpCas9-HF1 contains all four mutations). Although...substitutions throughout the groove in 32 separate Cas9 mutants. When tested for their ability to cut the EMX1 ...embryonic kidney (HEK) cells, 11 of these initial mutants retained the on-target activity of wt Cas9 while...locations in HEK cells. Further testing of two of the mutants, SpCas9(K855A) and eSpCas9(1.1), followed by a ...double strand breaks (BLESS), revealed that these mutants do not cause off-target effects at unanticipated...

...ways. Zebrafish mutants In the 1990’s, two revolutionary large-scale random mutagenesis screens produced.... These mutants are used extensively to study development and disease. Additional mutagenesis projects... on mutants and other resources. Targeted gene deactivation tools for zebrafish Random mutagenesis screens...developmental defects in the one-eyed pinhead mutant from the random mutagenesis screens. Microscopy-based lineage...produced ~1,500 zebrafish mutants that are still used in labs across the globe (Nüsslein-Volhard et al., 2012...which I will come back to) is the one-eyed pinhead mutant, which regulates a developmental pathway implicated...have since been initiated and today thousands of mutations in genes affecting diverse biological processes—not...

...Optimal mutation in-depth One the most sophisticated features of CrispyCrunch is how it mutates guide sequences...region, the mutation may cause side-effects. Further, work by John Doench et al. shows that mutating a single... After each mutation, it will check the CFD score. If it is below 0.03, it stops mutating. (Note: the ... example, it ranks guides by CFD score, and it mutates HDR donor templates to avoid re-cutting when needed...for TagIn. HDR requires the additional step of mutating guide sequences in a way that prevents re-cutting...do it best. Scientists are typically advised to mutate the PAM to which the guide binds. However, if the...implemented the following algorithm for optimal mutation in CrispyCrunch: If the guide PAM resides outside...

...Cancer Mutant Collection) or “pan-cancer” analyses across many cancer types (Pan Cancer Mutant Collection... cutting edge genomic technologies to map the mutations that are present, and use prior knowledge (data...One central bottleneck is that most recurrently mutated cancer genes are rare and most of the individual...no large scale, broadly available collection of mutant cDNA clones corresponding to cancer variants found...through Addgene. These include 634 sequence confirmed mutant cDNA clones corresponding to cancer variants derived...High-throughput Phenotyping of Lung Cancer Somatic Mutations. Berger AH, Brooks AN, Wu X, et al. Cancer Cell..., 2016. PubMed PMID: 27478040. The lung cancer mutant collection is described in this paper. Systematic...

...models with mutations in human-relevant exons 45-55, rather than in the traditional exon 23-mutated mouse model...modification, to repair or add in disease-causing mutations, but, until recently it hadn’t been used to treat... males. DMD is caused primarily by frameshift mutations in dystrophin, a protein essential for proper ...sequence appears to be nonessential. Within the mutational hotspot for dystrophin, exons 45-55, there are...partially functional protein. Individuals with these mutations are often asymptomatic, or have only mild symptoms... oligonucleotide exon skipping (OEN) to remove mutated exons from the dystrophin transcript. Unfortunately...which would prevent the accumulation of unwanted mutations over time. DMD is a good choice for a CRISPR proof...

...approaches. What is Rett Syndrome? Caused by a mutation in the methyl-CpG binding protein 2 (MECP2) gene...Zoghbi's lab discovered the first disease-causing mutations in the MECP2 gene leading to Rett Syndrome (Amir... regulate transcription (Baker et al., 2013). Mutations that affect this interaction to varying degrees...determine the clinical severity of different MECP2 mutations. More recently, the Bird lab found that only a... human MECP2 locus and MECP2 protein, showing mutations corrected by CRISPR/Cas9 in wild-type human iPSCs...CRISPR/Cas9 and RNA-mediated correction of MECP2 mutant alleles, in particular, have shown exciting and...and promising results for repairing mutations in models of neurological disease. Klaus Rajewsky’s group...

...multiple point mutations NEBuilder HiFi can simplify multi-position site-directed mutagenesis by assembling...primers with your desired mutation, performing PCR to generate the mutation and linearizing your vector... limit to where the mutation can be placed. With other techniques, when mutations are spaced farther apart...multiple synthetic fragments, create multiple mutations in one or multiple fragments, and generate constructs... Generation of constructs with multiple point mutations Cloning sgRNAs Production of Yeast Expression ...assembling multiple fragments containing the desired mutations. This same process can be used simultaneously ...simultaneously for the generation of one or more mutations – it is as easy as obtaining a plasmid with the gene of ...

...variants or mutations have been reported in people, we can use this kit to knock in those mutations. Once that's...efficiently generate human stem cells edited to carry mutant versions of 15 different disease-associated genes...homology-directed repair by flooding the cell with a mutant template and fooling the cell into using that template... you can either knock out a gene or knock in a mutation. Addgene: How do you use this kit to target a ... done in stem cells, we can differentiate those mutant and wild type stem cells into any cell type relevant...adipocytes or neurons, for instance – to see how those mutations influence the disease process. In the Cell Stem...The TALEN kit is quite useful in generating the mutant cell lines that one needs to do disease modeling...

...expressing plasmid to generate a population of mutant cells. The mutant cells were treated with WNV at a dose ...well as a dominant-negative mutator allele of the E. coli MMR protein MutL, all under the control of the...The temperature-controlled expression of the MutL mutant enables transient suppression of DNA repair during...sufficient to kill all control cells and surviving “mutant” colonies were deep sequenced to identify the gRNAs...differentiation, proliferation, and apoptosis. In humans, mutations that permanently activate Ras are found in one...vivo expression, and/or produce vectors to make mutant cell lines or transgenic animals.   Wall, et al...MAGE in otherwise unmodified bacterial strains (5). MutL is highly conserved in distant relatives of E. coli...

... and their mutations relative to wildtype GFP. Although not listed here, many permutations within each... and the mutation definitions found in Table 2 may not apply. Table 1: The specific mutations comprising...2: Functional role of specific mutations in GFP derivatives Mutation Known Function S65T Increased...little truncation is tolerated, however, point mutations are acceptable. GFP's main advantage over conventional...began engineering new versions of GFP through mutagenesis in order to improve its physical and biochemical...In 1995, Roger Y. Tsien described an S65T point mutation that increased the fluorescence intensity and ...facilitating its widespread use in research. Many other mutations have since been introduced to GFP and new iterations...

...biotin ligase, BirA*, with a site mutation (R11G). This site mutation destabilizes the ligase, allowing...evolution to generate two mutants, the 35 kD TurboID, which has 15 mutations compared to BirA and the ...compared to the standard E. coli BirA* mutant. RapID consists of this mutant BirA* and an RNA component that ...the E. coli version. It also contains a single mutation (R40G) in a conserved residue in the biotin catalytic...rounds of directed evolution until they identified a mutant with significantly increased sensitivity that did...original APEX (Lam et al. 2014). They named this mutant APEX2.  Find the APEX2 plasmids! TurboID and minturbo...which has the N-terminus domain deleted and 13 mutations compared to wild type BirA. TurboID and miniTurbo...

...phosphomimetic dual mutant) elevated kinase activity over 70-fold over dual phosphomimetic mutation. This emphasizes...phosphoserine-incorporation mutant for a residue that wasn’t originally a serine (e.g. mutating a threonine residue...residues. The phosphorylated residue of interest is mutated to a negatively charged residue, aspartate or glutamate...charge in the phosphate group. Additionally, these mutations are not reversible, and thus cannot reflect the...made constitutively active through phosphomimetic mutation of these residues. Due to the limitations previously...previously mentioned, phosphomimetic mutations on the activation loop do not fully activate MEK1. In fact...recombinant proteins as an alternative to phosphomimetic mutation. Creating phosphoserine-incorporated proteins ...

...engineered a dominant-negative mutant of the protein MLH1, a component of the MutSα–MutLα mismatch repair (MMR)...likely recognized as an MMR intermediate, and the MutSα–MutLα complex may either selectively excise the edited... PE6e-g editors with mutations to the Cas9 domain but found that these mutations only improved efficiency...created and evaluated 19 PE1 variants with RT mutations known to increase activity, enhance binding between...came out on top? The Cas9 nickase fused to a pentamutant of M-MLV RT. They called this system PE2, which...additional nuclear localization sequences; and two mutations in Cas9 previously shown to improve nuclease activity...effective prime editor proteins, but found that the mutations that emerged depended strongly on the target edit...

...heterogeneous population of mutant cells, with each cell or set of cells containing a mutation in a different gene...example, mutant cells can be used in drug screens to identify genes that confer drug-resistance. Mutant cells...library plus Cas9 to generate the mutant cells, respectively (C). Mutant cells are screened (D) and "hits... (NHEJ), usually results in a loss-of-function mutation within the target gene. CRISPR can also be used...experiment is to generate and screen a population of mutant cells to identify genes involved in a particular...system in which Cas9 must be delivered separately.  Mutant cells are enriched using either drug selection ...correspond to genes that confer drug resistance when mutated. Findings from this type of experiment can shed...

...dominant mutation, known as co-conversion (Arribere et al., 2014). In both cases, the selected mutation must... of lethal mutation co-selection. Only animals rescued for the conditional lethal mutation survive the...Cas9 allows us to delete sequences and introduce mutations and epitopes with unprecedented ease. These new...fluorescence (GFP; Tzur et al., 2013), or rescue of mutant phenotypes (unc-119) (Dickinson et al., 2013). ...approach relying on repair of a conditional-lethal mutation to identify edits with minimal screening effort...effort. Selecting for repair of a lethal mutation should remove much of the unedited “haystack”, facilitating...selection for repair of a temperature-sensitive pha-1 mutation significantly enriches for knock-in of 2x and ...

...frameshift mutation. The majority of cellular repair events will generate these types of mutations, making...recognition sequence is destroyed via mutation. By introducing a mutation in either the PAM or the guide recognition...mishaps in this class of genome edit. Knock-in mutations and gene editing Targeted genome editing events... this is not the case for knock-ins. Knock-in mutations typically require an exact DNA sequence to be ...cells. It is important to know that HR is often mutated in cancer cells, specifically in breast and ovarian...recommended you check your cell lines for HR-related mutations before attempting knock-ins by HDR.   ... with some success (Yu et al., Nat Chem Biol). Mutate the PAM or sgRNA sequences Cas9 likely goes through...

...with an Arabidopsis mutant that can’t set seeds. I study the development of the mutant plants, but the seeds...unfamiliar with these mutant plants. Using RUBY to simplify the identification of these mutants will speed up ...containing the desired mutation but lacking both RUBY and the construct that rescues the mutation. I will be able...identified by a phenotype caused by the segregating mutation. Selecting these plants is a learned skill that...up my work and make it easier to study these mutants.   Fig. 2: (A) Betacyanin is produced from ...do this by expressing a gene that rescues the mutations, using RUBY as a selection marker. These plants...because they will no longer show the effects of the mutation. But ¼ of the offspring from these plants will...

...could be used to edit several autosomal-dominant mutations that have been connected to the early onset of... CRISPR/Cas9 corrects Alzheimer’s disease mutation in basal forebrain cholinergic neurons derived...Academy of Sciences found that mice with a dominant mutation in a gene that causes cataracts could be rescued... Cas9 mRNA and a single-guide RNA targeting the mutant allele (7). An independent team led by Hans Clevers...demonstration of use of genome editing to correct point mutations in two diseases with real phenotypic effect.” ...noted that CRISPR/Cas9 has been used to generate mutations in a wide range of organisms, but its potential...phase of crisis where they 'collect' a number of mutations.” Cystic fibrosis may not be the most likely candidate...

.... J. (2008). Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following...discovered by researchers investigating HIV integrase mutants in the early 1990s (Leavitt et al., 1996), and ...lentiviral vector: 1. No risk of insertional mutagenesis Lentiviral vectors tend to integrate into transcriptionally...2002), thus increasing the risk for insertional mutagenesis. This integration could lead to an undesirable...harbor these unwanted interruptions. Insertional mutagenesis is especially concerning in clinical settings...acute lymphoblastic leukemia due to insertional mutagenesis (Howe et al., 2008). 2. Transient expression ...cells, as they avoid the risk of insertional mutagenesis while still allowing for stable expression of...

...astounding number of ways to create chromosomal mutations in bacteria, so many that it may be difficult ...path to take. A quick and easy way to introduce a mutation in the chromosome is to disrupt expression of ...genes. However, there are ways to create scarless mutations, ones that don’t leave any undesired scars in ...technology has also aided in the creation of scarless mutations, both in stand-alone systems (Jiang et al., 2013...plasmid curing steps, and can generate off-target mutations. Modifying bacterial genomes using counter-selectable...is frequently called allelic exchange. Here, a mutated allele introduced from a conjugative plasmid replaces...versatile technique, and is less prone to off-target mutations. Ultimately, successful allelic exchange requires...

...different mutations in a gene called SOX10. To understand the functional role of those mutations, they now...Syndrome, a devastating genetic disease caused by mutations in the OCRL1 gene that leads to the early death...to work to explore the effect of various OCRL1 mutations on cell behavior in greater detail. The goal is...CRISPR-Cas9 constructs from Addgene to gene-edit mutations into XP genes and other genes involved in photosensitivity...plasmids to explore the functional differences among mutations responsible for the disease known as Alternating...the brain. McSweeney says they now think that mutations in the gene may prevent neurons from returning...work will help them to understand how different mutations lead to differences in the symptoms experienced...

... Validating knockout mutations When a knockout cell line is desired, any mutation that introduces a frameshift...silent mutation in a neighboring nucleotide along with your desired edit. This silent mutation should ...cells were appropriately edited for your desired mutation - insertion, deletion, or site-directed knock-...is. If you are trying to introduce a homozygous mutation, it’s helpful to know how many copies you are ...Secondly, categorize your edit – is it any frameshift mutation? A specific insertion or deletion? A knock-in?...large deletion events.   Validating knock-in mutations You’ve introduced a donor template along with ... editor. What next? When generating a knock-in mutation, the best screening method depends on what your...

.... For example, mutations in the RBE site reduce binding with Rep proteins and mutations in the trs site...integrity and prevent unwanted mutations?  During propagation, mutants rapidly replicate in a growth culture...important for viral genome replication, these mutations can affect transduction efficiency and subsequent...are prone to replication errors and disruptive mutations and truncations that can make vector propagation...result in a high percentage of plasmid DNA with mutated ITR sequences. Using recombinant-deficient bacterial... sequencing is also helpful in quantifying ITR mutants since analysis of individual reads allows for identification...within the ITR. However, small deletions and point mutations will go undetected by restriction digest methods...

...for site directed mutagenesis. During this process, incorporation of a desired mutation into your plasmid...only the non-mutated and methylated template is destroyed leaving behind a pool of mutated plasmids which...Troubleshoot Your Sequencing Results Use REPLACR Mutagenesis to Mutate Your Plasmids Resources on Addgene.org ...newly synthesized product from a site-directed mutagenesis experiment? The answer to both questions is the...plasmid of interest by PCR generates mutated plasmids with no methylation (there are no methyltransferases...dam-/dcm- strains may have an increased rate of mutation (as these would also be deficient in mis-match...

...incredibly useful for generating mutations by NHEJ and generating small mutations with HDR, when it comes to...technologies, a researcher would first introduce a mutation into a mouse embryonic stem cell (ESC) line, enrich...that had successfully incorporated the desired mutation, and then derive mice from these engineered ESCs...the chromatogram showing the telltale signs of a mutation introduced by CRISPR. The sequence started clean...done, 75% of the mice in this experiment showed a mutation! I looked out my window and I saw the Dorr Mountain... many, if not all, the mice carry a frameshift mutation. For knock in with a donor oligonucleotide, we...successful in deriving mice carrying the intended mutation. With a donor plasmid, the outcome is less predictable...

...complexity using existing C. elegans mutants. For instance, we use a mutant known as CX51 that exhibits motor...effects this mutation has on locomotion in our computer model to the effects of this mutation in the real...real mutant worm. Like many academic labs, we have the capability to study many different mutants because...educational institution meaning that we can obtain known mutants at $7.00 a strain. Makerspaces like ours can also...to modify the worm genome and make additional mutations in our worms.  We are on the lookout for genome...model  so that we can use CRISPR/cas9 to build the mutants, analyse their motor behaviour, and ultimately ...

... track of your mutation. To maintain stable lines of flies carrying a specific mutation, Drosophila geneticists...whether stocks of mutant flies exist, whether any phenotypes have been observed from mutations in the gene,...third post will describe how you can make your own mutant flies. Find Drosophila Resources at Addgene The...contributed by mother and father). While some mutations can remain stable as homozygous alleles, others...method for maintaining stable lines of genetic mutants, including in situations where maintaining heterozygosity...you can stably maintain a heterozygote, and the mutation won’t drift away with each passing generation....journey of trying to get males to recombine two mutations onto the same chromosome. You should use females...

...pedal of a vehicle, respectively. In the case of a mutation that turns a proto-oncogene permanently “on”, ...accelerator of a vehicle being stuck down. Similarly, a mutation that turns a tumor suppressor “off” is analogous...From proteins to pathways - Ras as a model When mutations do arise and cancer ensues, traditional chemotherapies...proteins are special because they are known to be mutated in as many as 30% of all cancers, especially those...pancreas and colon. Despite the prevalence of Ras mutations in a variety of cancers, Ras proteins have stumped...approaching the Ras problem by exploiting the different mutant versions of Ras that are most prevalent in human...synthesizing compounds to bind specifically to their mutated residues. For Ras researchers, one of the most ...

...flies that contain a specific loss-of-function mutation in groucho have thick tufts of sensory bristles...embryo depending on its concentration. Genetic mutations in SHH cause severe health conditions such as ...reconstructed a transposase system based on the naturally mutated and non-functional transposase gene found in salmonid...name but some of our Addgenies had an idea: sqh mutant fly ovaries had “squiggly” actin fibers that resemble...spermatocytes to enter meiosis. Testis of cookie monster mutant flies are filled with early mitotic cells as well... who discovered comr thought the sperm cells in mutant flies looked like a “whole bunch of cookie monster...of this gene name but the larval gut of bagpipe mutants vaguely resemble a bagpipe and historically, bagpipes...

...rely on that clone not having any confounding mutations, the risk is high. You are generating a gene therapy...patients and the diseases associated with an elevated mutation burden. The CRISPR platform must be optimized ...hardly any flexibility in gRNA placement (e.g., mutating a specific amino acid or introducing a specified...evoCas9 are a few spCas9s which have been evolved or mutated to be more specific for exact gRNA matches (Rabinowitz...occurs. 2 gRNA approach Off-targets that come from mutations arise from base/prime editing or Cas cleavage....cleavage. In the case of Cas cleavage, off-target mutations can be mitigated by using two gRNAs in close proximity...the event required for frameshift and knock-in mutations. If one nickase acts on an off-target site, it...