GenCRISPR Services

Overview

As a leader in gene synthesis and genome editing, and through our partnership with Feng Zhang's laboratory at the Broad Institute of MIT and Harvard, GenScript offers comprehensive CRISPR solutions for reliable genome engineering, We are simplifying gene editing with user-friendly online design tools, useful protocols, and expert technical support to help you harness the power of CRISPR genome editing for your research.

CRISPR Basics

• What is CRISPR?

  CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) sequences were originally identified in the Escherichia coli (E. coli) genome, and were found to function as part of an RNA-based adaptive immune system to target and destroy genetic parasites at the DNA level.

  CRISPR systems have since been adapted for use in various eukaryotic cell types as a simple but versatile genome editing tool, allowing researchers to make specific changes to a cell’s DNA.

• CRISPR Components

  The most basic CRISPR gene editing experiment, gene knockout, requires two components to be delivered to the cell’s nucleus; a targeted guide RNA and a Cas nuclease. These components form a complex that binds DNA at the target sequence and generates a double-strand break (DSB), creating an opportunity for knockout.

  Guide RNA

  CRISPR guide RNAs are designed to target specific DNA sequences near a protospacer adjacent motif (PAM) site and may be used as single guide RNA (sgRNA) or as a duplex of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).

  GenCRISPR guide RNAs are available as synthetic sgRNA or plasmid crRNA.

  

  Cas Nuclease

  CRISPR associated protein (Cas) are RNA-guided nucleases capable of cleaving the phosphodiester bonds of a DNA backbone to create a double-strand break. The most commonly used Cas for CRISPR engineering is Cas9 from S. pyogenes (SpCas9). Cas9 orthologs have also been discovered from S. aureus (SaCas9) and C. jejuni (CjCas9). Additional naturally occurring Cas nucleases include Cas12a, Cas12c, Cas12e, Cas13a, and CasX. Variations in PAM specificity among these Cas variants will affect guide design flexibility and the potential for incidental off-target editing.

  In addition to the wild-type Cas nucleases, researchers have engineered variants for specific uses, including improved editing specificity (eSpCas9) and relaxed PAM specificity (SpCas9-NG). Researchers have also altered Cas9 nuclease activity for single-strand cutting (nCas9) or deactivated it entirely (dCas9) for use with transcriptional activators or repressors. For more information on these alternative CRISPR-Cas systems, please see the CRISPR Toolbox section below.

  GenCRISPR Cas Nucleases are available as protein or mRNA.

  Delivery

  Both CRISPR guide RNA and Cas nuclease must be delivered into the cell nucleus for successful editing to occur. CRISPR/Cas delivery strategies may be separated into two types; non-viral transfection and viral vector transduction. The chosen strategy will largely determine which reagent formats are suitable. For more information, see the Engineering Workflows section.

  Non-viral transfection	Viral vector transduction
  Deliver CRISPR components as RNA or protein directly into cell nucleus	Deliver virus packaged plasmids encoding CRISPR components for integration
  Electroporation, lipofection, microinjection, biolistics	Lentivirus,  AAV

  Multiple delivery strategies may also be used in series, for example by first transducing lentiviral packaged Cas9 plasmid to generate a stable Cas9 expressing cell line, and then electroporating guide RNAs for editing.

  Editing & Repair

  Once Cas9 nucleases are guided to the target DNA and create a DSB 3-4 bases upstream from the PAM sequences, there are two ways the DSB can be repaired.

  If there is no donor DNA present, resolution will occur by error-prone non-homologous end joining (NHEJ), resulting in an indel formation that effectively knocks out protein function in some percentage of the cell population.

  Alternatively, if donor DNA sequences are available, the DSB is repaired by homology directed repair (HDR) for precise knock-in of the target gene.

  
• Engineering Workflows

  Engineering Workflows

  The most common CRISPR engineering workflows include:

  Workflow	sgRNA	Cas	Recommended Delivery	Description	Ideal for	Benefits
  RNP	synthetic	protein	Transfection (electroporation, lipofection)	Synthetic CRISPR sgRNA and Cas protein are complexed as ribonucleoprotein (RNP) prior to delivery.	ex vivo editing, embryonic microinjection	DNA-free / transgene-free. Editing begins immediately.
  mRNA	synthetic	mRNA	Co-transfection	Synthetic CRISPR sgRNA and Cas mRNA are co-delivered.	in vivo editing	DNA-free / transgene-free. Editing begins post-Cas9 expression.
  Co-plasmid	plasmid	plasmid	Viral packaging	Separate viral packaged plasmids containing CRISPR sgRNA and Cas are co-delivered, and randomly integrate for constitutive expression.	Difficult-to-transfect cell lines	Simple delivery and selection/enrichment.
  All-in-one	single plasmid	single plasmid	Viral packaging	Single viral packaged plasmid containing CRISPR sgRNA and Cas is delivered, and randomly integrates for constitutive expression.	Difficult-to-transfect cell lines	Single viral transduction delivery step
  Stable Cas9 cell line	synthetic	plasmid	Viral Cas plasmid, transfected sgRNA	Viral packaged Cas plasmid is delivered to generate stable Cas-expressing cell line. Then synthetic sgRNA is delivered for editing.	Screening	Easily test stable Cas9 expressing line with various gRNA

• Theory to Action: Applying CRISPR in Research

  Theory to Action: Applying CRISPR in Research

  Traditional Gene Knockout

  As described above, the CRISPR-Cas complex generates a targeted double-strand break, creating an opportunity for gene editing.

  Gene knockout (KO) experiments exploit flaws in the cell’s native repair mechanism, non-homologous end joining (NHEJ). Imperfectly repaired genes with insertions or deletions of base pairs will result in frameshift mutations, rendering the gene and its corresponding protein non-functional. KO guides are algorithmically designed and ranked for both specificity (low homology with any other genomic sequence) and functionality (targeting a portion of the gene most likely to result in functional protein KO). CRISPR KO can be applied in many research scenarios, from basic screening, functional studies of a gene, bioengineering, agricultural biotechnology, to developing therapeutic gene and cell therapeutic drugs.

  Increasing CRISPR/Cas9 Editing Specificity

  CRISPR Cas9 based gene knockout relies on the specificity of both the sgRNA and the Cas9 protein. Poorly designed sgRNA and traditional wildtype Cas9 would lead to editing at undesired genome locations, which are considered as off-target edits. Many Cas9 variants have been developed for enhanced specificity. For example, the eSpCas9(1.1), also referred to as SpCas9 (K848A/K1003A/R1060A), structurally engineered from Feng Zhang lab contains alanine mutations that weaken the bounding between the HNH/RuvC groove and the none targeting DNA strand, reducing off-target effects by over 10-fold while maintaining robust on-target genome editing efficiency (Slaymaker et al. 2016).

  Researchers have also engineered Cas9 variants to “nick” a single DNA strand instead of creating double stranded DNA breaks. Two individual nickases targeting opposite DNA strands are necessary to generate a break site for gene editing, which significantly increases targeting specificity. For example, SpCas9 nickase (Cas9n D10A) contains a mutation allowing the endonuclease to create single-strand nicks, as opposed to DSBs. Pairing two opposite facing gRNA sequences with SpCas9 nickase can be an efficient method of gene editing that prevents unwanted indels from forming.

  

  Novel Editing Technologies Without DSBs (Prime Editing/Base Editing)

  Cas9 nickases also could enable novel single-strand CRISPR gene editing techniques that avoid the potential for unintended genomic changes posed by double-strand DNA breaks. Prime editing utilizes a Cas nickase protein fused to a reverse transcriptase (RT) to write a new sequence into a target DNA site directly. Prime editing guide RNAs (pegRNA) contain both a DNA targeting sequence and an RT template so that the pegRNA-dCas9 nickase complex can identify the target site, cut a single strand, and write the new sequence. Multiple versions of the prime editing system have been developed to improve editing efficiency.

  Base editing utilizes a Cas9 nickase, or alternatively a catalytically dead Cas9 (dCas9) which only binds to target DNA without cutting, fused to a nucleobase deaminase enzyme and a DNA glycosylase inhibitor to make targeted point mutations. For example, cytosine base editors (CBEs) and adenine base editors (ABEs), by fusing Cas9 nickase or dCas9 to a cytidine deaminase like APOBEC or adenine DNA deaminases, can convert C to T (or G to A) and A to G (or T to C).

  

  Traditional Gene Knockin

  Gene knock-ins have various applications in biotechnology, including disease modeling and gene and cell therapy applications. CRISPR based gene insertion relies on a pathway different than NHEJ, it is called Homology directed repair (HDR) pathway. HDR process is very precise allowing for accurate gene knockin, but HDR occurs in less frequency compared to NHEJ leading to less editing efficiency compared to knockout. KI experiments also require the introduction of a Donor DNA HDR templates, which could be either single- or double-stranded DNA designed with overlapping homology arms to the specific cut site.

  To increase the efficiency of HDR, experiment optimization by testing different template format, delivery approaches, culture conditions, as well as using small molecules for cell cycle synchronization, enhance HDR or inhibit NHEJ have been used to overcome this challenge.

  Read more on HDR template formats available and design.

  

  CRISPR Transcriptional Regulation

  Additional Cas9 variants have been developed for transcriptional regulation of gene expression. Rather than editing DNA, these deactivated Cas9 proteins simply bind the target site and activate or inhibit expression.

  CRISPR activation (CRISPRa) systems utilize a dCas9 fused to transcriptional activators to up regulate endogenous gene expression, whereas CRISPR interference (CRISPRi) systems use dCas9 fused to transcriptional repressors to downregulate gene expression. Multiple CRISPRa and CRISPRi systems have been developed using different activators or repressors. CRISPRa gRNAs for the SAM system are currently available in plasmid formats.

  Researchers have also recently developed a Cas fusion protein, CRISPRoff, that can epigentically modify and silence genes without editing the underlying genome. The epigenetic memory persists through cell differentiation and is heritably passed to future cell generations. Gene activity can be re-activated using a different fusion protein called CRISPRon.

  Technique	Components Required	Common Experiments	Common Applications
  CRISPRa	CRISPRa guide RNA + dCas9-SAM (or other)	Overexpression screening and hit analysis	Drug discovery
  CRISPRi	CRISPRi guide RNA + dCas9-KRAB (or other)	Transient loss-of-function, siRNA validation	Drug discovery
  CRISPRoff/ CRISPRon	CRISPRoff guide RNA + CRISPRoff	Heritable gene silencing without DNA editing	Gene therapy

  
• Should You Consider Other Cas Protein Options?

  Should You Consider Other Cas Protein Options?

  Though Cas9 protein has been widely popular and used in various research projects, did you know that there are many other types of Cas proteins that might be even better suited for your specific research needs?

  CRISPR/Cas systems can be categorized into two classes, Class I and Class II, based on differences in the organizations of effector modules. Research suggests that all CRISPR/Cas systems may have originated from a common ancestor. In this evolutionary process, class I systems were encoded by a single-function cas gene and lost some additional cas genes over time, forming class II systems. These classes are further divided into six types: type I to type VI. Class I includes type I, III, and IV, while class II includes type II, V, and VI (Table 1). Different types of CRISPR/Cas systems have different functions based on the structure and function of their Cas proteins. Type I, II, and V systems are involved in recognizing and cleaving DNA, while type VI can edit RNA. Type III has the ability to edit both DNA and RNA. As for the effect of type IV systems on DNA or RNA, it is still not fully understood and requires further study. By understanding the different classes and types of CRISPR/Cas systems, we can better appreciate the diversity and potential applications of this technology. Here we will highlight Cas12 and Cas13 as the two other commonly used Cas protein types used in research now than Cas9.

  Class	Type	Effector/Protein	Target Molecule	Need tracrRNA	PAM/PFS
  1	I	Cascade (e.g. Cas3)	DNA	No	-
  	III	Cascade (e.g.Cas10)	DNA/RNA?	No	-
  	IV	Cascade (Csf1)	DNA	No	-
  2	II	Cas9	DNA	Yes	SpCas9 & FnCas9 uses ′NGG’
  	V	Cas12	DNA	Yes/No	TTN or TTTN
  	VI	Cas13	RNA	No	A, U, or C

  Table 1: Summarization of commonly used Cas protein types and characteristics.

  Cas12

  There are 3 subtypes under Cas12, including Cas12a, Cas12b, and Cas12c, among which Cas12a is the most popular. Cas12a, as known as Cpf1, distinguishes itself from Cas9 through several key characteristics:

  • Cas12a is a compact and efficient enzyme with size ~1,300 amino acids, thus easier to package for therapeutic delivery using adeno-associated virus (AAV), which has limited capacity.
  • Cas12a possesses a single RuvC endonuclease active site, creating 5' overhangs sticky ends instead of blunt ends at the cut site, making it more convenient for applications like HDR based editing.
  • Cas12a demonstrates a preference for a 5' protospacer adjacent motif (PAM), which allows for efficient targeting of AT-rich genomic loci without the need for G's or C's in the PAM sequence.
  • Cas12a does not require a tracrRNA and only need a short crRNA to function, which is easier and cheaper to synthesize compared to long single guide RNA required by Cas9.
  • One notable feature of Cas12a is its ability to indiscriminately cleave single-stranded DNA once activated, making Cas12a a powerful tool for detecting trace amounts of target DNA in diagnostic applications.

  Cas13

  Unless Cas9 and Cas12, Cas13 family is specific for targeting RNA sequences. Currently, there have been four subtypes of Cas13 that have been characterized: Cas13a, Cas13b, Cas13c, and Cas13d. Cas13 protein sizes range from ~900-1250 amino acids, and Cas13 guide RNA consists of a conserved hairpin-like structure of around 70 nucleotides and a variable region of 29 nucleotides that is complementary to the target RNA. Once activated, Cas13 exhibits nonspecific RNase acidity and cleaves all nearby RNA molecules regardless of sequence, which is leveraged in diagnostic tool development, for example Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK). In addition, Cas13 is also used widely for RNA knockdown in eukaryotic cells, as well as RNA tracking/imaging and modification via using an catalytically inactive Cas13 (dCas13) which only bind, but not cleave RNA.

  
• How to Design & Pick the Best gRNA?

  How to Design & Pick the Best gRNA?

  Guide RNAs are the foundation of any CRISPR-based gene editing experiment, therefore the design of gRNAs is a critical step in ensuring successful outcomes. To maximize the efficiency of edits and avoid off-target effects, careful considerations should be given to the following key areas in gRNA design:

  1. Efficiency and Specificity: A high-performing gRNA should effectively target the desired DNA sequence with high specificity. Mismatches between the gRNA and the target site can result in off-target effects, depending on the number and position of the mismatches. Minimizing off-target effects is crucial to ensure the accuracy of gene editing experiments and prevent unintended mutations in other genomic regions.
  2. Target Accessibility: Some genomic regions may pose challenges for effective targeting. Understanding the accessibility of the target site helps prioritize gRNAs that are more likely to lead to successful edits. In certain cases, designing multiple gRNAs for each gene of interest may be necessary due to unpredictable activity and specificity.
  3. Leverage Free Online Design Tools: Various online tools are available that can assist in gRNA design by predicting efficiency, specificity, and potential off-target effects. These tools utilize algorithms and databases to optimize gRNA selection. GenScript also offers a free GenCRISPR design tool that can help you select gRNA for gene knockout applications.
  4. Choose Ideal gRNA Format: gRNA can be delivered using plasmids or virus containing gRNA encoding sequence, synthetic crRNA:tracrRNA system, IVT sgRNA or synthetic sgRNA. Among these, synthetic sgRNA with modifications grants the best editing efficiency and minimum immunogenicity in cells for in vivo animal applications. Consider the experimental requirements and choose the most appropriate gRNA format, purity grades and modifications if applicable, accordingly.