...double-stranded template.  Using viral genome sequencing to verify the viral genome We hypothesized that this double...the time and cost for AAV genome sequencing.  Figure 1: The viral genome sequencing workflow. ... and created a simplified process, termed viral genome sequencing (VGS), to confirm the identity and serotype... NGS is an effective tool to confirm AAV vector genome identity. These studies used SMRT sequencing, TA-based...ligation and tagmentation-based methods for AAV genome sequencing (Tai 2019; Lecomte 2015; Maynard 2019...step was assumed to be necessary because the AAV genome is single-stranded and both TA-based ligation and... might not actually be necessary. Since the AAV genome exists as [+] and [-] strands that are packaged...

...desired genomic target provided that (1) the sequence is unique compared to the rest of the genome and (...cleaving genomic DNA in mammalian cells and zebrafish embryos, and they can be used to modify genomic loci... is an intrinsically high-fidelity genome-editing platform. Genome Biol 19: . https://doi.org/10.1186/...Cas9 (3'-NGG) are abundant throughout the human genome, they are not always positioned correctly to target...sequence may have high homology elsewhere in the genome. These off-target sequences may be unintentionally...PAM. This variant may increase the specificity of genome modifications at DNA targets adjacent to NGG PAM...targeting range of CRISPR/Cas9 within the human genome. Another variant, xCas9 3.7 has 7 mutations found...

...CasX Drug inducible CRISPR/Cas9 activation E. coli genome-wide inhibition library CRISPR knockout libraries...Cas9 generates a double-stranded DNA break at the genomic loci of interest and on either side of the cargo...either side of the double-stranded break in the genomic loci of interest. As the universal sgRNA is not...to target sites in the human, pig, or zebrafish genome, this system can be used in these organisms. Read... Drug inducible transcriptional activation and genome editing Yu Wang's lab developed drug inducible ...Cas9 systems for transcriptional activation and genome editing by fusing Cas9 to a mutated human estrogen... Find the drug inducible Cas9 plasmids E. coli genome-wide inhibition library In the last edition of ...

...modify the desired genomic locus.   Some historical notes CRISPR arrays in bacterial genomes consist of repeated...26, 2020. At their most basic level, CRISPR/Cas9 genome editing systems use a non-specific endonuclease...endonuclease (Cas9 or closely related Cpf1) to cut the genome and a small RNA (gRNA) to guide this nuclease to a...required for endonuclease function, when used for genome engineering, the only CRISPR protein required is...more detail below, enables Cas9 to cut a specific genomic locus of many possible loci. Without binding to... (PAM) Cas9 endonuclease binding to the target genomic locus is mediated both by the target sequence contained...requirements allow researchers to target many different genomic loci. 3. Cleave target DNA resulting in a double-strand...

... AAV-based genome editing In 2016, Jonathan Ploski’s lab developed a viral mediated genome editing system...for your research.     CRISPR-Cas12b for human genome editing The best studied Cas12b nuclease from Alicyclobacillus...cleavage at 48 °C, a temperature incompatible to genome editing in human cells. The Feng Zhang lab identified...or plants of rice and wheat. Read the paper in Genome Biology Find plasmids containing these base editors...Staphylococcus aureus Cas9 (SaCas9). This AAV-based genome editing system is regulated not only temporally...Molecular Neuroscience Find the AAV-based SaCas9 genome editing plasmids String assembly gRNA cloning...various environments. It has the advantage of stable genomic integration and is easily transferred via conjugation...

...to mutate in the genome) and an out-of-frame H2B (histone)-GFP reporter into the genome of mammalian cells...If you’re using CRISPR to make a genome edit, how do you know if your CRISPR experiment was successful...experiment and confirm the correct edit was made to the genome without any off target effects. However, these ...for the reporter to work and to make the desired genome edit:  A donor plasmid that contains a puromycin...GFP reporter leaving you with just your desired genomic edit.  Find SRIRACCHA plasmids at Addgene GEMCherry2...activity Guide RNA structure and the site in the genome you are trying to target play a large role in Cas9... lab first incorporated an out-of-frame CRISPR genomic target region into the N-terminus of mCherry. To...

...sit down with Wei Leong Chew, a researcher at the Genome Institute of Singapore who recently started his... Chew's career path and why he chose to go into genome engineering. Chapter 1: Early research on CRISPR...graduate work to an independent position at the Genome Institute of Singapore. Chapter 3: Biology research...projects. Chapter 6: Projects in Wei Leong Chew's lab. Genome engineering, gene delivery methods, AAVs, and more... Chew is a Junior Principal Investigator at the Genome Institute of Singapore. His lab focuses on producing...producing biotechnologies in the areas of genome editing, immunology, and gene therapy.   Special thanks... genome engineering...

... “default” genome looks like, which led to the incorporation of ancestral bias into genome-wide screens...and the human genome Wang, T., Antonacci-Fulton, L., Howe, K. et al. The Human Pangenome Project: a global...digging deeply into their data.     Fig. 1: Genomic fraction for each of five major ancestry groups...ancestry-associated genes.   What does the reference genome actually reference?  The CRISPR guides used in ...designed to have a perfect match to the reference genome – even single mismatches within a CRISPR guide ...guide can prevent the guide from binding to the genome or prevent Cas9 from cutting, both of which can introduce...significant bias into the results. But the reference genome does not represent the diversity of genetic variation...

...of the genome, which seem resistant to Cas9 cleavage (1). Both the CRISPR-Cas9 and NgAgo genome engineering...AND FOLLOW UP STUDIES HAVE FAILED TO DEMONSTRATE GENOME EDITING BY THIS TOOL This post was contributed ...but does not perform functional tests. The newest genome engineer sharing the stage with much-lauded CRISPR-Cas9...remember that NgAgo has only been available for genome editing for a few months. More time is required...NGG may not be present in AT-rich regions of the genome or just after a stop codon (as required for C-terminus...Researchers have been excitedly testing NgAgo's genome editing applications. So, how does NgAgo stack ...the respondents have been struggling to achieve genome editing with NgAgo, this does not mean the technology...

...insert new DNA at the target genomic loci.  A last word on the depiction of genome engineering in pop culture... be designed each time we wanted to target new genomic sites. The discovery of the Cas proteins in 2005...predecessor induce a double-strand break at the target genomic location. This is typically followed by non-homologous... they induce double-strand breaks (DSBs) in the genome. These technologies rely on the cellular DNA repair...lead to high frequency of indel formation in the genome and are inefficient at introducing point mutations...new strategy to introduce point mutations in the genome. They fused a catalytically impaired Cas9 protein...introduce a C→T change at precise locations in the genome. This was soon followed by the adenine base editor...

...virtually any genomic locus, provided the target is unique compared to the rest of the genome and located...CRISPR-based reagents are available for genome-wide screens? The goal of a genome-wide screening experiment is to...gene in the genome, or a specific class of genes? Addgene currently carries several genome-wide CRISPR... how CRISPR libraries are being used to perform genome-wide screens and highlight some of the reagents... A major advantage of CRISPR/Cas9 over previous genome editing techniques is its simplicity and versatility... target genes without permanently modifying the genome. If you'd like to brush up on the various CRISPR...gRNA-containing plasmids, each with a different genomic target. The goal of a CRISPR screen is to use Cas9...

...compare the genomes of these two family members.   Figure 1: Gamma-retrovirus genome (left) and ...in the vector (or are nearby in the host genome). The genome of viral tools are often not virology related...Cullen, 1992) because of their more complicated genomes. Here, we will mostly use the gamma-retroviruses...and an HIV lentiviral genome (right.)  HIV seems an unlikely candidate for use as a tool in the lab ...present in the capsid, as the engineered, recombinant genome is (thankfully) gutted of many essential viral ...the vector particle, along with its RT-deficient genome. In both cases, the lipid membrane is the only ...this reverse-transcribed DNA into the host cell’s genome and then use cell machinery for transcription and...

...elements capable of spreading within and between genomes, and have been adapted for a wide variety of applications...involved, they insert either randomly, or at fixed genomic target sites.  This obstacle was recently surpassed... insertions of large DNA cargos into bacterial genomes. This new capability was named INTEGRATE (Insertion...TniQ for targeting (Halpin-Healy et al., 2020). Genomic targets require an upstream 2-bp PAM sequence for...Guided by the crRNA, QCascade complexes survey the genome and bind target DNA sequences using RNA-DNA base...molecule, and inserts it ~50 bp downstream of the genomic target site bound by QCascade (Fig. 1B). The process... al., 2020).  CRISPR-transposons for bacterial genomes engineering In the Sternberg group’s initial report...

...cis-acting signals for genome replication and viral gene transcription (6). The EBOV genome contains seven viral...the viral genome and transcribes viral genes. Four viral proteins are required for viral genome replication...of the minigenome to produce the positive sense, genome replicative intermediate (anti-minigenome). The...under BSL-2 conditions (4). What is a minigenome?   The EBOV genome is a nonsegmented, negative sense RNA... sequence (gene end). A minigenome is a shortened version of the viral genome. It contains the 3’ and ...the negative sense minigenome, thus modeling genome replication. These products can be detected by Northern...studied by changing the minigenome cis-acting sequences and monitoring effects on genome replication and transcription...

...block? The Cas family of proteins has many useful genome engineering enzymes, each with their own unique...unique features and quirks. Here, we will review the genome engineering abilities of the lesser-known Cas enzymes...to Cas9. DNA targeting Cas enzymes Cas9 – The genome engineering workhorse You rarely hear the word ...thereafter hearing ‘Cas9’. The Cas9 nuclease is a genome engineering powerhouse, and for good reason. It...molecule, the gRNA, to a complementary site in the genome where it will proceed to generate a blunt DNA double... double strand break. Cas9 has been studied for genome engineering purposes far more than any other Cas...sites, making Cas9 an adaptable choice for many genome engineering endeavors. For more information on ...

...of scientists to make genomic alterations, bringing about a revolution in genome engineering, with new... protein and a guide RNA (gRNA) targeting your genomic site of interest. This is commonly performed by...       Plasmid DNA may be incorporated into the genome     Off-target effects can occur due to prolonged...of interest. Cas9 RNPs are capable of cleaving genomic targets with similar efficiency as compared to ...Cas9/gRNA and can be used for most of the current genome engineering applications of CRISPR including: generating...homology directed repair (HDR), and generating large genomic deletions. What are the advantages of Cas9 RNPs...long-term expression of Cas9, such as visualizing genomic loci using fluorophore tagged dCas9 may require...

...plant genome editing. Cytosine base editors (CBE) Precise and efficient base rewriting in the genome became...one genomic site simultaneously.     These binary vectors could be used to generate stable genome edited...cellular polymerases. Subsequently, an original genomic A-T base pair is converted to a G-C pair (Figure...protein like UGI in CBE. Base editors for the plant genome These preliminary studies demonstrated successful...vectors to install A>G or T>C mutations in the plant genome. We constructed three versions of the vector (pKABE6.3...ABE7.10 can edit target 'A' if it lies in a small genomic window: at protospacer position 4-8 counting the...do not find a suitable ‘NGG’ PAM at your target genomic locus/loci, choose a base editor variant from Table...

...albicans E. coli genome-wide inhibition library To expand the tools available for genome-wide studies in...CRISPR tools for your research! This time: E. coli genome-wide CRISPR inhibition Covalent tethering of DNA...in E. coli, the Chong Zhang lab developed a genome-scale CRISPR inhibition pooled library (~60,000 gRNAs... without any significant hit across the E. coli genome as a negative control. Read the paper in Nature...Nature Communications Find the E. coli genome-wide CRISPR inhibition library Covalent tethering of DNA...HDR) can be used to make precise modification in genome editing. However, HDR efficiency is low. Wendy ...cytosine base editor (CBE) with rat APOBEC1 causes genome-wide deamination of RNA cytosines in human cells...

...specific genomic loci (CRISPR-imaging), or to find the proteins associated with a given genomic locus (...enriches for knock-outs and knock-ins at other genomic loci. The first approach – co-CRISPR from Craig...Recent work has further improved the efficiency of genome editing by CRISPR whether it be by improved gRNA...et al., 2017). Because C. elegans has an AT-rich genome, it can be difficult to find an appropriate NGG...allows editing from more sites of the C. elegans genome. Toolkits for editing in C. elegans The development.../Cas Toolkit and SapTrap-SEC kit for C. elegans genome engineering have also facilitated CRISPR editing... novice researchers to quickly and easily start genome editing (Schwartz et al., 2018). To see more CRISPR...

... what if you want to study genes all across the genome? Two new human lentiviral CRISPR library systems...Institute's Ophir Shalem. “Whole regions of the genome which were not accessible before are now accessible...accessible using this technology.” GeCKO library for genomic screens Using lentivirus as the delivery vehicle..., cell cycle proteins, and more.  “In the past, genome engineering with CRISPRs was limited to modification...hand, uses a 1 plasmid, single infection system. Genome wide mouse CRISPR library The Yusa lab has leveraged...-Cas9 can be used to target any sequence in the genome. Tips and tricks Wang offers this tip: “It's quite...DM, Lander ES. Science. 2013 Dec 12. (PubMed). Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells...