LbCas12a

Cas ID

RESOURCES

CLASSIFICATION

  • Cas ID 3.4.3
  • Nuclease Activity Target dsDNA (or ssDNA) + trans-ssDNA activity. Staggered cut.
  • Targeting Requirement 5' PAM
  • gRNA and Multiplexability crRNA
  • Class 2 subtype V-A
  • PAM or PFS TTTV

PROPERTIES

  • Protospacer Length 23 1 2
  • PAM TTTV 3
  • Protein Weight (KDa) 143.7
  • RNP Weight (KDa) 157.4
  • CDS Length (nt) 3684 3
  • Number Amino Acids 1228 3
  • tracrRNA Length (nt) 3
  • crRNA_Length (nt) 20 1 2
  • sgRNA Length (nt) 3



DESCRIPTION

Summary

LbCas12a (formerly LbCpf1) is an RNA-guided, DNA-targeting CRISPR-Cas nuclease identified from Lachnospiraceae bacterium 3 . The RNA-guided double-stranded DNA endonuclease activity of LbCas12a was experimentally characterized in 2015, alongside that of other Type V-A/Cas12a orthologs such as AsCas12a and FnCas12a 3 . Like other Cas12a orthologs, LbCas12a catalyzes sequence-specific cleavage of RNA-complementary double-stranded DNA (cis cleavage), as well as indiscriminate cleavage of single-stranded DNA (trans cleavage) at a single active site in the RuvC domain 4 3 5 . LbCas12a is one of the CRISPR-Cas12a endonucleases to be subsequently established as a staple in the CRISPR toolbox 6 7 . Compared to AsCas12a, which is another Cas12a ortholog used in gene-editing applications, LbCas12a has been shown to be more active at a lower range of temperatures from 22°C to 37°C in vitro and in cells 8 9 10 .

Applications

The RNA-guided endonuclease activity of LbCas12a has been harnessed for biotechnological applications in healthcare, agriculture, and fundamental research 11 12 13 6 14 . Due to LbCas12a’s increased activity at temperatures below 37°C compared to other Cas12a orthologs, LbCas12a has been utilized for editing in plants 8 9 10 . Some genome-editing applications include increasing tomato salinity tolerance 15 and gene replacement for herbicide resistance in rice at Cas12 target sites |reference_idx|, but several examples may be found in the experimental section of CasPEDIA.

Experimental Considerations

When utilizing LbCas12a and other Cas12a orthologs, such as AsCas12a, MbCas12a and FnCas12a, one should take into account several experimental considerations. Multiple software tools, such as CHOPCHOP 16 and CRISPOR 17 , exist for guide design 18 to help identify suitable target sites throughout the genomes of several model organisms. LbCas12a recognizes a 5'-TTTV-3' PAM on the non-target strand of dsDNA 3 . LbCas12a shares a preference for T-rich PAMs with other Cas12a orthologs, unlike Cas9a’s preference for G-rich PAM sequences 19 . Staggered cuts generated by Cas12a have been reported to result in larger deletions than those of SpyCas9 |reference_idx|20 21 . Furthermore, Cas12a’s ability to process its own crRNA has enabled multiplexed editing with multiple crRNAs in a single RNA transcript 22 23 . When designing LbCas12a’s crRNA scaffold sequence, one should consider the promoter and model organism. To accommodate transcription initiation at RNA polymerase III (Pol III) or T7 promoters, an additional single or dinucleotide ‘G’ can be appended to the 5’ end of the crRNA scaffold sequence reported in LbCas12a’s sequences section 24 13 . Increased editing efficiencies have also been reported with pre-crRNAs compared to mature crRNAs in rice, and crRNAs with additional 5’ nucleotides have been designed for Cas12a processing 25 24 . Variants of LbCas12a have been engineered for novel PAM-targeting specificities, activity at a wider range of temperatures, and enhanced on-target nuclease activity 26 27 28 26 29 .

Search Constructs on Addgeneaddgene_link

Tool_Type Tool Is_Tool_Applicable Notes Citation
NaN RNP Yes Established to work as WT and with variants with improved nuclease activity 30
NaN Lenti Yes Activity profiling of LbCas12a and AsCas12a 19
NaN AAV Yes LbCas12a and crRNA was packaged within an AAV serotype 9 vector and adminstered to mouse eye by intravitreal injection 12
NaN LNP Yes Delivery of crRNA and mRNA into mice livers 13
NaN EDV Yes In principle, but not yet demonstrated NaN
NaN Guide_Design_Algorithm NaN CRISPOR; CHOPCHOP guide design algorithims 17 16
NaN Other_Experimental_Notes NaN Can produce multiple gRNA on one promoter to facilitate easy targeting of multiple loci. 23

Application_Type Description Pharmaceutical_or_Product_Name NCT Responsible_Party Delivery_Mechanism In_Vivo_or_Ex_Vivo_Editing Citation_or_Publications
Plant_Agriculture LbCas12a genome editing in Nicotiana benthamiana, Solanum lycopersicum, Arabidopsis thaliana NaN NaN NaN Stable transformation of Arabidopsis thaliana by Agrobacterium tumefaciens In vivo 31
Plant_Agriculture LbCas12a-mediated genome editing in soybean and tobacco protoplasts NaN NaN NaN PEG-mediated RNP delivery to soybean and tobacco protoplasts Ex vivo 10
Mouse LbCas12a targeting of the PCSK9 gene in mouse hepatocytes to reduce of serum cholesterol levels NaN NaN NaN LbCas12a RNP delivery with a DNA nanoclew (NC)–based carrier. LbCas12a RNP-NC was additionally coated with a cationic layer of polyethyleneimine (PEI) as well as an anionic polymer layer. In vivo 13
Mouse LbCas12a editing of Vegfa or Hif1a in mouse retinal and retinal pigment epithelium (RPE) cells. LbCas12a editing was demonstrated to reduce the area of laser-induced choroidal neovascularization. NaN NaN NaN AAV serotype 9 vector, adminstered to the mouse eye by intravitreal injection In vivo 12
Yeast Multiplexed genome editing by LbCas12a in S. cerevsisiae, where three genes of the heterologous carotenoid pathway were integrated into three different genomic loci NaN NaN NaN Chemical transformation of S. cerevisiae NaN 32

Tool_Type Tool_Name Description Citation
Nucleic acid detection diagnostic NaN DNA endonuclease-targeted CRISPR trans reporter (DETECTR): Attomolar sensitivity DNA detection; Detection of Severe fever with thrombocytopenia syndrome virus (SFTSV) infection; Detection of Cancer associated hotspots 4 33 34
Transcriptional repression dLbCpf1–SRDX dLbCpf1 (D832A) was fused to three copies of the SRDX transcriptional repressor, and reported to repress expression of the non-coding RNA miR159b in Arabidopsis. 35
Adenine base editor LbABE8e Catalytically dead LbCas12a was fused to an evolved version of Escherichia coli tRNA deaminase (a monomeric TadA-8e variant). LbCas12a-mediated adenine base editing was demonstrated at target sites with TTTV PAMs 36 , as well as target sites with noncanonical PAMS when TadA-8e was fused to LbCas12a with evolved PAM recognition preferences 35 . 36 35

Variant_Name Description
Lb2-KY (C1003Y, Q571K) An engineered variant of LbCas12a with improved nuclease activity and broadened PAM targeting (CTTN) 37
LbCas12a-RV(G146R/R182V), LbCas12a-RRV (G146R, D156R, E795Q), and LbCas12a-RVQ (G146R/R182V/E795Q) Engineered LbCas12a variants were demonstrated to have enhanced editing efficiencies in human cells (LbCas12a-RVQ), rice and tomato protoplasts (LbCas12a-RV), and stably transformed rice and poplar plants (LbCas12a-RRV) 38
LbCas12a RVRR (G532R, K538V, Y542R, K595R), impLbCas12a (D156R, G532R, K538V, Y542R, K595R) Broadened PAM recognition ((TAYV, TGTV TTCV, CCCV and TCCV) was demonstrated by the LbCas12a RVRR variant. However, the LbCas12a RVRR variant was demonstrated to cleave at more off-target sites than WT LbCas12a. "Improved" impLbCas12a (LbCas12a RVRR with an additional D156R mutation), is reported to to have enhanced activity and a further broadened PAM targeting specificity to include NTTV PAMs. Additional LbCas12a variants with altered PAM recognition preferences were reported. 27
LbCas12a-Plus (D156R, R883K, R887A) LbCas12a variant demonstrated to have enhanced activity and specificity 26
ttLbCas12a (D156R) A "temperature-tolerant (tt)" LbCas12a variant which demonstrated increased editing activity in Arabidopsis thaliana from 22°C and 28°C 28
mut2B-W (K623R, F863V, Q1108L, S1132T, S1214P), mut2C-W (F863V, F884L, D952N, C965Y, V1011A, Q1108L, A1113V, S1132T, S1214P), and mut2C-WF (F863V, D952N, C965Y, V1011A, Q1108L, A1113V, S1132T, S1214P) Hyper-effective (HypE) LbCas12a variants with enhanced endonuclease activity, demonstrated in vitro and in genome editing with HEK293T cells. These variants are hypothesized to adopt a more open conformation 29

NUCLEOTIDE SEQUENCE


PROTEIN STRUCTURE

PFAM ID Description
PF18510 NUC
PF18501 REC1
PF18516 RuvC_1

Feature Type Start End Ligand Description Citations
Domain 49 280 Cas12a REC1
Domain 810 1225 Cas12a RuvC nuclease
Domain 1004 1166 Cas12a nuclease
Region 47 51 Binds crRNA alone and in crRNA-target DNA heteroduplex
Region 154 158 Binds crRNA alone and in crRNA-target DNA heteroduplex
Region 256 260 Binds DNA in crRNA-target DNA heteroduplex
Region 278 281 Binds crRNA in crRNA-target DNA heteroduplex
Region 516 520 Binds crRNA
Region 707 710 Binds crRNA
Region 719 720 Binds crRNA
Region 781 789 Binds crRNA
Coiled coil 77 107
Active site 759 759 For pre-crRNA processing
Active site 768 768 For pre-crRNA processing
Active site 785 785 For pre-crRNA processing
Active site 832 832 For DNase activity of RuvC domain
Active site 925 925 For DNase activity of RuvC domain
Active site 1180 1180 For DNase activity of RuvC domain
Site 16 16 Binds crRNA alone and in crRNA-target DNA heteroduplex
Site 272 272 Binds DNA in crRNA-target DNA heteroduplex
Site 286 286 Binds DNA in crRNA-target DNA heteroduplex
Site 514 514 Binds DNA in crRNA-target DNA heteroduplex
Site 538 538 Binds DNA protospacer adjacent motif (PAM)
Site 591 591 Binds Target strand DNA
Site 595 595 Binds PAM
Site 601 601 Binds Target strand DNA
Site 740 740 Binds Target strand DNA; via amide nitrogen
Site 747 747 Binds crRNA

PDB_IDs Domains Active_Sites
5ID6 RuvC D850;E943;D1198

PDB ID: 5XUS


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