CRISPR Overview

Site-specific nucleases, and among them, CRISPR/Cas are powerful tools for genome engineering. CRISPR/Cas was adapted from an immune system in bacteria and requires two components: a guide RNA (sgRNA) and a CRISPR associated endonuclease (Cas protein). The efficiency, versatility and multiplexing capacity of this technology enables a variety of applications in plants, ranging from gene knockouts to homologous recombination or precise base editing. Most of the applications depend on the technical ability to compose and transfer complex synthetic instructions into the plant cell.

In order to simplify the assembly of constructs for gene editing in plants, we adapted the CRISPR/Cas9 and the CRISPR/Cas12a technologies to the GoldenBraid cloning platform. You can read more about gene editing with Cas9 here and about gene editing with Cas12a here.

Which Cas should I choose for gene editing?

Cas9 and Cas12a (previously named Cpf1) are both endonucleases of a Class 2 CRISPR-Cas system. Cas9 is naturally a dual-guided endonuclease but the crRNA and tracrRNA, that exist as two separate RNA molecules in nature, were engineered in a single RNA molecule (sgRNA) that has become the most popular format for CRISPR guide RNAs. Cas12a is a single-guided endonuclease and therefore its natural crRNA is used by researchers in the same format. Althouhg the election of the Cas is case-dependent, some differences among them that might affect the decission are the PAM sequence or their (in)ability to process polycistronic transcripts. The table below summarizes the main differences among the two nucleases.

How to assemble single gRNAs for Cas9 with GB?

1) Adapt your target sequence to the GB standard here. Dicot and monocot target sequences should start with 'G' and 'A' respectively. This constraint derives from the RNA polymerase III-dependent U6 and U3 promoters requirement of a 'G' or 'A' at the 5' end of the RNA to be efficiently transcribed.

2) Assemble your GB-adapted target sequence with a PolIII promoter and the SpCas9 scaffold to create a guide RNA transcriptional unit here.

3) Combine the TU created at 2) with extra single or polycistronic gRNAs and our recommended TUs/modules for gene editing using the binary assembler.

How to assemble polycistronic gRNAs for Cas9 with GB?

1) Decide the number of gRNAs that you want to assemble in a single polycistron. These polycistronic RNA will be processed in planta by the endogenous tRNA processing system (RNase P and RNase Z) producing individual functional guide RNAs (tRNA-based strategy). See Xie et al. for details on the use of gRNA-tRNA arrays for multiplex genome editing.

2) Adapt your target sequences to the GB standard here. Number of target sequences to adapt to the GB standard will depend on the number of total gRNAs you want to include in the polycistron.

3) Clone in the pUPD2 a tRNA-protospacer-scaffold unit for each position in the polycistron. For example, for a 4X polycistronic RNA you will need four tRNA-protospacer-scaffold units cloned in the pUPD2, each of them with a different part category (E1, E2, E3-E4-En-1, EnC1). To do so click here.

4) Create a TU with a PolIII promoter and the previously assembled tRNA-protospacer-scaffold units here.

5) Combine the TU created at 4) with extra single or polycistronic gRNAs and our recommended TUs/modules for gene editing using the binary assembler.

How to assemble single crRNAs for Cas12a with GB?

1) Adapt your target sequence to the GB standard here. GB accepts Cas12a target sequences ranging from 20 to 23 nts.

2) Assemble your GB-adapted target sequence with a PolIII promoter that already includes the Cas12a direct repeat sequence here. Notice that this transcriptional unit also includes a HDV 3' processing signal.

3) Combine the TU created at 2) with extra single or polycistronic crRNAs and our recommended TUs/modules for gene editing using the binary assembler.

How to assemble polycistronic crRNAs for LbCas12a with GB?

Note: so far gbcloning only supports assemblies of polycistronic crRNAs for LbCas12a.

1) Decide the number of crRNAs that you want to assemble in a single polycistron. Gbcloning supports asselmblies of polycistrons with up to 6 crRNAs. Cas12a has endoribonuclease activity for processing its own crRNAs.

2) Select the most suitable option depending on the number of crRNAs you want to assemble here and upload the target sequences. GB accepts Cas12a target sequences ranging from 20 to 23 nts. Target sequences will be assembled in the final construct in the same order you type them here.

3) Order the synthesis of the crRNA-array you will get as result of 2). We recommend ordering it at GenScript as a Gene Synthesis product and clone it in the pUPD2.

4) Assemble the polycistronic crRNA together with the U6-26-LbCas12aDR promoter fusion here.

5) Combine the TU created at 4) with extra single or polycistronic crRNAs and our recommended TUs/modules for gene editing using the binary assembler.