Full stack genome engineering: the end-to-end solutions making crispr accessible to all

CRISPR is the coolest technology in a scientist’s toolbox these days because of its unique blend of being extremely powerful yet relatively simple to use. The technique relies on two basic

components: a CRISPR-associated (Cas) nuclease that cuts DNA and a guide RNA (gRNA) that tells the nuclease precisely where in the genome to cut. The CRISPR system enables scientists to make

changes virtually anywhere in the genome of any organism. The possibilities of such a technology are vast, from curing genetic diseases to making crops resistant to pests. Given the immense

potential of CRISPR, scientists all across the world are excited to start using it in their research. Once researchers have familiarized themselves with the basics of CRISPR, the next step

is to start editing genomes in their own experiments. However, the path to becoming a genome engineer is not as straightforward as one might think. For instance, scientists planning to use

CRISPR will need to ask themselves the following: _What is the best way to design my gRNA?_ _Which nuclease is appropriate for my experiment?_ _Is one transfection method better than the

other?_ _How do I choose the best CRISPR analysis tool? _ All these decisions can be overwhelming, and it’s sometimes hard to even know where to start. GETTING STARTED WITH CRISPR – THE EASY

WAY To make all this a bit simpler, let’s consider the entire CRISPR experiment subdivided into three broad steps: Design → Edit → Analyze. In short, scientists must first _design_ their

gRNA, then _edit _the genome, and lastly _analyze_ their results. This holistic view of the CRISPR experiment is what we call FULL STACK GENOME ENGINEERING. STEP 1: DESIGN THE CRISPR GUIDE

RNA AND SELECT THE CAS NUCLEASE The first step in your CRISPR experiment is to design a gRNA to target your DNA sequence. The editing efficiency and specificity of the guide RNA will greatly

influence the success of your CRISPR experiment. There are different formats and ways of making guide RNAs. The guide RNA format of choice for many of the top genome engineers is synthetic

single guide RNAs (sgRNAs) that are chemically modified so that they persist longer within cells. The design of your gRNA also depends on the nuclease you intend to use, as each gRNA must

bind near a nuclease-specific PAM sequence. Although Cas9 is a currently the most popular Cas, a variety of alternative nucleases exist. Therefore, it is worth doing some research on the

appropriate CRISPR nuclease depending on the application and specific experimental needs. STEP 2: EDIT DNA PRECISELY WITH CRISPR After all CRISPR components have been selected, one more

decision remains. What is the best delivery system to introduce the CRISPR components into the cell? There are several options for transfecting cells, all which have different advantages for

different cell types. Once a transfection method is chosen, you must optimize a multitude of conditions in order to ensure that a high amount of editing takes place in your cell type. This

can be a laborious process but is critical to achieving high editing efficiency. STEP 3: ANALYZE DATA FROM THE CRISPR EXPERIMENT You picked the best gRNA and used an optimum transfection

method, and now you need to analyze your CRISPR edits. Choosing a user-friendly and accurate analysis method is essential to understanding how well the editing worked and to determine the

next steps in your experiment. With your editing efficiency and knockout score known, you can now decide whether to perform assays on your pool of cells or perhaps continue to generate

clones. SYNTHEGO’S FULL STACK GENOME ENGINEERING APPROACH Now that we have walked through the Design → Edit → Analyze steps of the CRISPR workflow, we hope that you have a better idea of all

the work that goes into a CRISPR experiment. Synthego’s Full Stack Genome Engineering solutions encompass the entirety of CRISPR experiments. We provide comprehensive support for every step

of the genome engineering workflow, allowing all scientists to access CRISPR and advance their research. Our automated, full stack genome engineering platform enables broader access to

CRISPR to accelerate basic scientific discovery, uncover cures for diseases and develop novel synthetic biology applications. Learn more at Synthego.com.